The cloning of genes, gene fragments and other DNA sequences is a fundamental part of
molecular biology. To study the function of a particular DNA sequence, you must be able
to manipulate that sequence. There are two main ways to achieve this: the polymerase
chain reaction (PCR) and the more traditional use of restriction enzymes and modifying
enzymes to “cut and paste” the desired DNA fragments into cloning vectors, which can
then be replicated using live cells, most commonly E. coli. The use
of PCR has an advantage in that it gives you the option to re-amplify the target DNA
each time your DNA supplies dwindle without ligation into a vector or transformation
into E. coli. Alternatively, PCR products can be ligated into a
suitable vector, which can then be transformed into and replicated by E.
coli. This chapter covers the basics of cloning using PCR and restriction
enzymes, including DNA cleanup prior to ligation, ligation, transformation and screening
to identify recombinant clones.
The PCR process is a useful tool to quickly and easily amplify the desired sequences.
With the successful sequencing of whole and partial genomes of organisms across all
biological kingdoms, DNA cloning by PCR is an easily attainable option. Public DNA
databases allow researchers to design primers to amplify their DNA fragment of interest
directly from the genomic DNA of the desired organism. With the simple addition of a
reverse transcription step prior to PCR, RNA sequences can be converted to cDNA, which
can then be cloned into a suitable vector. For additional information about
amplification of DNA and RNA sequences using PCR, see the PCR
Amplification chapter of the Protocols and Applications
Guide.
PCR products generated using a nonproofreading DNA polymerase such as
Taq DNA polymerase, which lacks 3´?5´ exonuclease
activity, have a single template-
independent nucleotide at the 3´ end of each DNA strand (Clark, 1988; Newton and
Graham, 1994). This single-nucleotide overhang, which is most commonly an A residue,
allows hybridization with and cloning into T vectors, which have a complementary 3´
single T overhang. PCR products generated using a proofreading DNA polymerase, such as
Pfu DNA polymerase, have blunt ends and must be cloned into a
blunt-ended vector or need a single 3´A overhang added to ligate into a T vector (Knoche
and Kephart, 1999).
If PCR amplification of the desired DNA fragment is not possible or desirable,
restriction enzyme digestion of the target DNA can be employed. The desired fragment may
need to be separated from other DNA fragments in the reaction, so the size of the
desired DNA fragment should be known. Once isolated, the fragment is cloned into a
vector with compatible ends. If the vector ends are capable of relegating (e.g., the
vector has blunt ends or is cut with a single restriction enzyme), the vector is often
treated with alkaline phosphatase to discourage recircularization and maximize ligation
between the insert and vector.
Following transformation into E. coli, the resulting bacterial
colonies are screened by PCR for the correct recombinant vector using primers to amplify
the insert. Alternatively, the recombinant vector can be identified by performing a
restriction enzyme digestion to determine the presence of the correct insert. Screening
is often simplified by using vectors that contain an antibiotic-resistance gene, so
cells containing the vector will survive on medium supplemented with the appropriate
antibiotic. Screening can be further simplified by choosing a vector and E.
coli strain that are compatible with blue/white screening, which takes
advantage of intracistronic a-complementation to regenerate ß-galactosidase activity.
Many E. coli strains used for cloning and propagation of plasmids
contain a chromosomal deletion of the lac operon but carry an F´
episome that provides the remaining coding sequence of the lacZ
gene. The functional lacZ gene product, ß-galactosidase, is
produced when the lacZ coding information missing on the F´ episome
is provided by the plasmid. This activity is detected by plating bacteria transformed by
plasmids on plates containing isopropyl ß-D-thiogalactopyranoside
(IPTG; an inducer of the lac promoter) and
5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal; a dye that
produces a blue color when hydrolyzed by ß-galactosidase). When the reading frame of the
a peptide is disrupted by insertion of a foreign DNA fragment or deletion of
vector sequences, a-complementation does not occur, and the bacterial colonies remain
white or occasionally light blue.
return to top of page
The use of amplification enzymes is the first step in cloning by PCR. Most people
use PCR for cloning, taking advantage of the single nucleotide A overhang left after
amplification with a nonproofreading DNA polymerase to ligate the amplimer to a
vector containing T overhangs. However, products will be blunt-ended if the DNA
polymerase has 3'?5' exonuclease activity, also known as
proofreading activity. Alternatively, PCR primers can add sequences for restriction
enzyme sites, and the resulting products can be digested and ligated into a vector
with compatible ends. Promega provides several thermostable DNA polymerases. These
include the GoTaq® Amplification Family,
Tfl DNA Polymerase and proofreading polymerases. A detailed list
of the various enzymes for use in PCR can be found in the Protocol and
Applications Guide chapter on PCR
Amplification, in the section "Thermostable DNA Polymerases". The
GoTaq® Amplification Family of products is highlighted
in the following section.
GoTaq® Amplification Family
GoTaq® DNA Polymerase is available in various
formulations to suit your needs: the standard GoTaq®
DNA Polymerase, which is supplied with a 1X reaction buffer that contains 1.5mM
MgCl2; GoTaq® Flexi DNA
Polymerase, which allows a range of MgCl2 to be used for
PCR; GoTaq® Green Master Mix, which is a premixed,
ready-to-use solution containing GoTaq® DNA Polymerase,
dNTPs, MgCl2 and reaction buffer at optimal concentrations
for efficient amplification of DNA templates by PCR;
GoTaq® Hot Start Polymerase, which includes a
proprietary antibody that blocks polymerase activity until the initial
denaturation step; and GoTaq® Long PCR Master Mix,
which offers efficient amplification of long templates (e.g., human genomic DNA up
to 30kb). All GoTaq® PCR Core Systems offer complete
solutions with polymerase and nucleotides; the GoTaq®
PCR Core System II also includes a positive control.
GoTaq® products contain Taq DNA
polymerase in a proprietary formulation that offers enhanced amplification over
conventional Taq DNA polymerase. Each member of the
GoTaq® family has a reaction buffer that contains
two dyes (a blue dye and a yellow dye) that separate during electrophoresis to
show migration progress as well as a compound that increases sample density.
Samples can be loaded directly onto gels without the need to add a separate
loading dye. If the dyes interfere with your downstream applications,
GoTaq® DNA Polymerases are supplied with a 5X
Colorless Reaction Buffer. Alternatively, the PCR Master Mix offers a ready-to-use
formulation without any dyes. Reaction products generated with these systems
contain A overhangs and are ready for T-vector cloning. Alternatively, the PCR
product can be digested directly with a restriction enzyme that is active in the
PCR buffer and cloned into standard cloning vectors (see Technical Manual #TM367 for a protocol).
Additional Resources for GoTaq® DNA Polymerase
Promega Publications
PN083
Introducing GoTaq® DNA Polymerase:
Improved amplification with a choice of buffers
Citations
Ning, B.
et al. (2011) 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by
increasing c-myc nuclear localization and binding to the e-boxes of
transferrin receptor 1 (
TfR1) and ferrochelatase
(
Fech) genes.
J. Biol. Chem. 286, 37196–206.
The authors performed real-time PCR using
SYBR® Green and 1.25 units of
GoTaq® DNA Polymerase to amplify 20ng of
cDNA generated from total RNA extracted from murine erythroid leukemia
(MEL) cells and mouse erythroid burst-forming units (BFU-Es) in a
total reaction volume of 25µl.
PubMed Number:
21903580
Vucurovic, K.
et al. (2010) Serotonin 3A receptor subtype as an early and protracted marker of
cortical interneuron subpopulations.
Cereb. Cortex 20, 2333–47.
After reverse transcription, PCR was performed to simultaneously
detect mRNAs encoding two isoforms of glutamic acid decarboxylase,
three calcium-binding proteins, three neuropeptides, two transcription
factors and reelin, a protein thought to be involved in neuronal
migration and morphology. Two rounds of PCR using nested primers were
required to detect these mRNAs. PCRs were performed using
GoTaq® DNA Polymerase. Amplified
products were visualized by agarose gel electrophoresis, using the
100bp DNA Ladder as a size standard.
PubMed Number:
20083553
Additional Resources for GoTaq® Flexi DNA
Polymerase
Promega Publications
PN089
GoTaq® Flexi DNA Polymerase: Robust
performance with magnesium optimization
Citations
Westphal, A.
et al. (2011) General suppression of
Escherichia coli O157:H7
in sand-based dairy livestock bedding.
Appl. Environ. Microbiol. 77, 2113–21.
DNA was extracted from bedding material and the 16S rRNA genes
amplified in a 25µl reaction using 1.5 units
GoTaq® Flexi DNA Polymerase with 1.8mM
MgCl2. The PCR products then were cloned
into the pGEM®-T Easy Vector.
PubMed Number:
21257815
Additional Resources for GoTaq® Green Master Mix
Promega Publications
eNotes
Activity of Promega restriction enzymes in
GoTaq® Green Master Mix and PCR Master
Mix
eNotes
Analyses of gene disruption by whole-cell PCR using the
GoTaq® Green Master Mix
PN101
Recombinant clone screening using the
GoTaq® Hot Start Green Master Mix
Citations
Crawford, M.A.
et al. (2011) Identification of the bacterial protein FtsX as a unique target of
chemokine-mediated antimicrobial activity against
Bacillus
anthracis.
Proc. Natl. Acad. Sci. USA. 108, 17159–64.
To identify transposon insertion sites, bacterial genomic DNA was
isolated, digested and ligated with a partially double-stranded
Y-linker. An initial 20µl amplification for 20 cycles using
GoTaq® Green Master Mix enriched ssDNA
fragments. A second PCR amplified dsDNA, adding more
GoTaq® Green Master Mix for a final
reaction volume of 100µl with 25 cycles. The amplimers were analyzed
by sequencing.
PubMed Number:
21949405
Additional Resources for PCR Master Mix
Promega Publications
eNotes
Activity of Promega restriction enzymes in
GoTaq® Green Master Mix and PCR Master
Mix
Additional Resources for GoTaq® Core PCR Systems
Technical Bulletins and Manuals
TB254
GoTaq® PCR Core Systems Technical
Bulletin
Citations
Fuehrer, H.P.
et al. (2011) Novel nested direct PCR technique for malaria diagnosis using filter
paper samples.
J. Clin. Microbiol. 49, 1628–30.
The authors developed a direct-amplification, nested PCR protocol
to amplify Plasmodium DNA from S&S 903
filter paper punches containing whole blood. The
GoTaq® PCR Core System amplified 5µl of
template (extracted from paper punches and whole blood in parallel) in
the second nested reaction using 2mM MgCl2 and
1 unit of GoTaq® DNA polymerase in a total
reaction volume of 50µl.
PubMed Number:
21270224
Additional Resources for GoTaq® Hot Start
Polymerase
Technical Bulletins and Manuals
9PIM500
GoTaq® Hot Start Polymerase Product
Information
Citations
Li, Z.
et al. (2011) The barley
amo1 locus is tightly linked to the
starch synthase
IIIa gene and negatively regulates
expression of granule-bound starch synthetic genes.
J. Exp. Bot. 62, 5217–31.
To examine the mutations in class II and class III starch synthases
(ssIIa and ssIIIa, respectively), genomic DNA from young barley leaves
was extracted and 50ng amplified in a 20µl reaction that included 1.5U
of GoTaq® Hot Start Polymerase, 1.5mM
MgCl2 and the additives DMSO and betaine.
After 35 cycles, the PCR products were digested overnight using EcoRI
and separated on 2% agarose gels.
PubMed Number:
21813797
Additional Resources for GoTaq® Long PCR Master Mix
Technical Bulletins and Manuals
TM359
GoTaq® Long PCR Master Mix Technical Manual
T vectors are a specific type of cloning vector that get their name from the T
overhangs added to a linearized plasmid. These vectors take advantage of the A
overhangs on PCR products after amplification with Taq DNA
polymerase by providing compatible ends for ligation (Mezei and Storts, 1994; Robles
and Doers, 1994). There are three different T-cloning vectors from Promega: Two are
basic cloning vectors, and the third is a mammalian expression vector.
pGEM®-T and pGEM®-T Easy
Vector Systems
The pGEM®-T (Cat.# A3600,
A3610) and pGEM®-T Easy Vector Systems
(Cat.# A1360, A1380) are convenient systems for
cloning PCR products. The vectors are prepared by cutting with a restriction
endonuclease to leave a blunt end and adding a 3´terminal thymidine to both ends
(Figures 13.1 and 13.2). These single 3´ T overhangs at the insertion site greatly
improve ligation efficiency of a PCR product into the plasmid by preventing
recircularization of the vector and providing a compatible overhang for PCR
products with 5' A overhangs.
The high-copy-number pGEM®-T and
pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase
promoters flanking a multiple cloning region within the coding region for the
a-peptide of ß-galactosidase. Insertional inactivation of the a-peptide allows
recombinant clones to be directly identified by color screening on indicator
plates containing X-Gal (Cat.# V3941) and IPTG
(Cat.# V3955). Both the
pGEM®-T and pGEM®-T Easy
Vectors contain numerous restriction sites within the multiple cloning region. The
pGEM®-T Easy Vector multiple cloning region is
flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI,
thus providing three single-enzyme digestions for release of the insert. The
pGEM®-T Vector cloning region is flanked by
recognition sites for the enzyme BstZI. Alternatively, a double digestion may be
used to release the insert from either vector.
The pGEM®-T and pGEM®-T
Easy Vectors also contain the origin of replication of filamentous phage f1 for
the preparation of single-stranded DNA (ssDNA). Both
pGEM®-T vector systems include a 2X Rapid Ligation
Buffer for ligation of PCR products, which requires only a 1-hour incubation at
room temperature. The incubation period may be extended to increase the number of
colonies after transformation. Generally, an overnight incubation at 4°C will
produce the maximum number of transformants.
Inserts of several kilobases have been successfully cloned into the
pGEM®-T and pGEM®-T Easy
Vectors
(D’Avino et al. 2004). However, as the insert gets
larger, the ratio of vector to insert may need to be optimized further to maximize
ligation efficiency (see Ligation and
Transformation in the section "Vector:Insert Ratio").
One of the disadvantages of PCR cloning into a T vector is that the insert can
be cloned in either direction. Analysis of recombinant vectors by PCR or
restriction enzyme digestion can be used to determine not only the success of
cloning but also the insert orientation. To verify the direction of the insert,
amplify recombinant plasmids using one of the gene-specific PCR primers and one
of the phage promoter primers that are present on the
pGEM®-T Vector (Knoche and Kephart, 1999). The correct
orientation is important for transcription or translation or both.
Additional Resources for the pGEM®-T and
pGEM®-T Easy Vector Systems
Technical Bulletins and Manuals
TM042
pGEM®-T and
pGEM®-T Easy Vector Systems Technical
Bulletin
Promega Publications
eNotes
pGEM®-T Easy Vector System is an easy tool
for preparing gel shift probes
PN071
Cloning blunt-end Pfu DNA Polymerase-generated
PCR fragments into pGEM®-T Vector
Systems
Citations
Maruyama, A.
et al. (2011) The novel Nrf2-interacting factor KAP1 regulates susceptibility to
oxidative stress by promoting the Nrf2-mediated cytoprotective response.
Biochem. J. 436, 387–97.
A mouse KAP1 expression plasmid was constructed by amplifying the
KAP1 cDNA in three fragments from RNA isolated from NIH3T3 cells. Each
of the fragments were cloned into the
pGEM®-T Easy Vector. The three recombinant
vectors were digested with restriction enzymes (HindIII and BamHI;
BamHI; BamHI and XbaI) and the resulting fragments were ligated
together and subcloned into an expression vector.
PubMed Number:
21382013
Aquilini, E.
et al. (2010) Functional identification of the
Proteus
mirabilis core lipopolysaccharide biosynthesis genes
J. Bacteriol. 192, 4413–24.
To identify the core lipopolysaccharides (LPS) biosynthesis genes
in Proteus mirabilis, 11 genes from P.
mirabilis strain R110 and one from strain 51/57 were
amplified from chromosomal DNA, cloned into the
pGEM®-T Vector and transformed into DH5a
competent cells. Once the cloned genes were confirmed, each
recombinant plasmid was transformed into Klebsiella
pneumoniae core LPS mutants to see if any of the
P. mirabilis genes complemented the
mutants.
PubMed Number:
20622068
pTARGET™ Mammalian Expression Vector System
The pTARGET™ Mammalian Expression Vector System
(Cat.# A1410) is a convenient system to clone
PCR products and express cloned PCR products in mammalian cells. As with the
pGEM®-T and pGEM®-T Easy
Vector Systems, the pTARGET™ Vector is supplied already
linearized with single T overhangs (Figure 13.3). These single 3´ T overhangs at
the insertion site greatly improve the efficiency of ligation of a PCR product
into the plasmid. The pTARGET™ Vector also contains a
modified version of the coding sequence of the a peptide of ß-galactosidase, which
allows recombinants to be selected using blue/white screening.
The pTARGET™ Vector carries the human cytomegalovirus
(CMV) immediate-early enhancer/promoter region to promote constitutive expression
of cloned DNA inserts in mammalian cells. This vector also contains the neomycin
phosphotransferase gene, a selectable marker for mammalian cells. The
pTARGET™ Vector can be used for transient expression or
for stable expression by selecting transfected cells with the antibiotic G-418.
Like the pGEM®-T or pGEM®-T
Easy Vectors, inserts of several kilobases can be cloned in and expressed from the
pTARGET™ Vector (Sakakida et al.
2005;
Le Gall et al. 2003).
Additional Resources for the pTARGET™ Mammalian
Expression Vector System
Technical Bulletins and Manuals
TM044
pTARGET™ Mammalian Expression Vector System
Technical Manual
Promega Publications
PN082
Technically speaking: T-vector cloning
Citations
Dastidar, S.G., Landrieu, P.M. and D'Mello, S.R. (2011) FoxG1 promotes the survival of postmitotic neurons.
J. Neurosci. 31, 402–13.
Four FoxG1 deletion mutants were generated by PCR, and with an
added C-terminal Flag tag, cloned into the
pTARGET™ Mammalian Expression Vector. The
mutant constructs were transfected into neuronal cells and neuronal
survival assessed.
PubMed Number:
21228151
Carpenter, J.E.
et al. (2011) Autophagosome formation during varicella-zoster virus infection
following endoplasmic reticulum stress and the unfolded protein response.
J. Virol. 85, 9414–2.
Four varicella-zoster virus (VZV) major structural glycoproteins
open reading frames (ORFs) were amplified from cultured cells infected
with laboratory strain VZV-32 and ligated into the
pTARGET™ Mammalian Expression Vector. The
recombinant vectors were grown, purified and transfected into HeLa
cells at a concentration of 0.5 µg/ml. After a six-hour incubation,
the medium was changed and the cells observed under confocal
fluorescence microscopy up to 24 hours later for autophagosome
formation.
PubMed Number:
21752906
The Flexi® Vector Systems (Cat.# C8640,
C8820, C9320) are based on a simple, yet powerful, directional
cloning method for protein-coding sequences. First, a PCR product is generated using
primers designed with two rare-cutting restriction enzymes, SgfI and PmeI. After
restriction enzyme digestion, the insert is ligated in a single orientation. All
Flexi® Vectors carry the lethal barnase gene, which is
replaced by the DNA fragment of interest and acts as a positive selection for
successful ligation of the insert. The two restriction enzymes provide a rapid,
efficient and high-fidelity way to transfer protein-coding regions between a variety
of Flexi® Vectors without the need to resequence while
maintaining the reading frame (see
Figure 13.4 for a system overview and Figure 13.5 for a list of example
vectors). Find a current list of available vectors at: www.promega.com. To design PCR primers appropriate for your insert and
with SgfI and PmeI restriction sites, visit the Flexi® Vector Primer Design Tool.
Unlike site-specific recombination vector systems, the
Flexi® Vector Systems do not require appending multiple
amino acids to the amino or carboxy termini of the protein of interest (Figure 13.6).
In addition, the systems do not require an archival entry vector, and most
applications allow direct entry into the vector suited to the experimental design
(e.g., mammalian expression or N-terminal, glutathione-S-transferase (GST) fusion
vectors). For instance, you might clone your PCR product into the pFN2A (GST)
Flexi® Vector to express your GST-tagged protein in
E. coli for purification. However, an easy transfer of your
insert after SgfI/PmeI digest followed by ligation into the pF4K CMV
Flexi® Vector will allow you to transfect the same
protein-coding region into a mammalian cell and determine its expression level.
Any Flexi® Vector can act as an acceptor of a
protein-coding region flanked by SgfI and PmeI sites (Figure 13.7). The SgfI site is
upstream of the start codon of the protein-coding region, and depending upon the
Flexi® Vector used for cloning, this allows expression
of a native (untagged) protein or an amino (N)-terminal-tagged protein by readthrough
of the SgfI site. The PmeI site contains the stop codon for the protein-coding region
and appends a single valine residue to the carboxy (C)-terminus of the protein
(Figure 13.6).
The C-terminal Flexi® Vectors allow expression of
C-terminal-tagged proteins. While these vectors can accept protein-coding regions
flanked by SgfI and PmeI, they lack a PmeI site and contain a different blunt-ended
site, EcoICRI. Inserts cloned using these sites cannot be removed from the C-terminal
Flexi® Vectors and transferred to other
Flexi® Vectors (Figure 13.4, Panel B).
Additional Resources for the Flexi® Vector Systems
Technical Bulletins and Manuals
TM254
Flexi® Vector Systems Technical
Manual
Promega Publications
PN093
The Flexi® Vector Systems: The easy way to
clone
PN091
Metal affinity tag for protein expression and purification using the
Flexi® Vectors
Online Tools
Flexi® Vector Systems Overview Animation
Flexi® Vector Primer Design Tool
Citations
Kuhn, P.
et al. (2006) Automethylation of CARM1 allows coupling of transcription and mRNA
splicing.
Nucleic Acids Res. 39, 2717–2.
Full-length mouse coactivator-associated arginine methyltransferase 1
(CARM1) was amplified and cloned into the pFC14K
HaloTag® CMV Flexi®
Vector. An R551K mutation was created in the same vector. The
HaloTag® constructs were transfected into
HEK293T cells, the CARM1 proteins affinity purified using HaloLink™ Resin
and the CARM1 cleaved from the C-terminal
HaloTag® using TEV protease. The purified
CARM1 then was analyzed by mass spectrometry.
PubMed Number:
21138967
Markandeya, Y.S.
et al. (2011) Caveolin-3 regulates protein kinase A modulation of the
Ca
V3.2 (a1H) T-type
Ca
2+ channels.
J. Biol. Chem. 286, 2433–44.
Full-length and truncated aveolae containing scaffolding protein
caveolin-3 (Cav-3) were fused to gluathione-S-transferase (GST) by PCR,
Pme1 and Sgf1 digestion and ligation in the pFN2A (GST)
Flexi® Vector. After confirming the
Cav-3-GST fusion constructs, the vectors were transformed into
E. coli strain BL21(DE3) and protein expression
induced by IPTG and purified using MagneGST™ Glutathione Particles. After
elution, the Cav-3 proteins were analyzed using Western blotting.
PubMed Number:
21084288
Promega offers a vast array of both modifying enzymes (e.g., ligase or
phosphatase) and restriction endonucleases for use in cloning. This section is an
overview of the products available from Promega to enhance your cloning results and
highlights the enzymes that may be most useful to you. For example, ligase is a key
enzyme in cloning as this enzyme joins the vector and insert to create a circular
recombinant plasmid. Restriction enzymes (REs) are used to cut a vector and PCR
product, or other type of insert, to generate compatible ends for ligation. REs also
can be used to evaluate ligation success by screening the recombinant plasmid for the
correct restriction sites. To explore strategies for subcloning, visit the Subcloning
Notebook.
DNA Ligase
DNA ligase catalyzes the joining of two strands of DNA using the 5´-phosphate
and the 3´-hydroxyl groups of adjacent nucleotides in either a sticky-ended or
blunt-ended configuration (Engler and Richardson, 1982). This allows the "pasting"
together of inserts and receptive vectors (e.g., A-tailed product into T vectors).
T4 DNA Ligase (Cat.# M1801, M1804,
M1794) can join DNA strands together and has been shown to
catalyze the joining of RNA to a DNA or RNA strand in a duplex molecule. However,
DNA ligase will not join single-stranded nucleic acids (Engler and Richardson,
1982).
Additional Resources for T4 DNA Ligase
Technical Bulletins and Manuals
9PIM180
T4 DNA Ligase Promega Product Information
The LigaFast™ Rapid DNA Ligation System (Cat.#
M8221, M8225) is designed for efficient ligation of
sticky-ended DNA inserts into plasmid vectors in just 5 minutes (blunt-ended
inserts in as little as 15 minutes). Rapid ligation is based on the combination of
T4 DNA Ligase with a unique 2X Rapid Ligation Buffer. The LigaFast™ System
eliminates any further purification prior to transformation of ligated DNA. The
specially formulated 2X Rapid Ligation Buffer requires no additional ATP or
Mg2+.
Additional Resources for the LigaFast™ Rapid DNA Ligation System
Technical Bulletins and Manuals
9PIM822
LigaFast™ Rapid DNA Ligation System Promega Product
Information
Promega Publications
eNotes
Cloning differential display-PCR products with
pGEM®-T Easy Vector System
PN071
Rapid ligation for the pGEM®-T and
pGEM®-T Easy Vector Systems
Alkaline Phosphatases
Alkaline phosphatases catalyze dephosphorylation of 5´ phosphates from DNA.
These enzymes are used to prevent recircularization and religation of linearized
vector DNA by removing 5´-phosphate groups from both termini and also may be used
to dephosphorylate 5´ phosphorylated ends of DNA for subsequent labeling with
[32P]ATP and T4 Polynucleotide Kinase. Unit usage
guidelines are usually included with the alkaline phosphatase (e.g., 0.01 units
per picomole of ends). For assistance in calculating picomoles of vector or insert
ends for dephosphorylation, visit the BioMath Calculators.
TSAP Thermosensitive Alkaline Phosphatase (Cat.#
M9910) catalyzes the removal of 5´-phosphate groups from DNA and
is effective on 3´ overhangs, 5´ overhangs and blunt ends. TSAP is active in all
Promega restriction enzyme buffers, a convenience that allows a single,
streamlined restriction enzyme digestion-dephosphorylation step. TSAP also is
inactivated effectively and irreversibly by heating at 74°C for 15 minutes.
Therefore, a DNA cleanup step is not required before ligation.
Additional Resources for TSAP Thermosensitive Alkaline Phosphatase
Technical Bulletins and Manuals
9PIM991
TSAP Thermosensitive Alkaline Phosphatase Promega Product
Information
Promega Publications
eNotes
TSAP Thermosensitive Alkaline Phosphatase activity in restriction
enzyme buffers from New England Biolabs
PN095
TSAP: A new thermosensitive alkaline phosphatase
Alkaline Phosphatase, Calf Intestinal (CIAP; Cat.#
M1821, M2825), catalyzes the hydrolysis of 5´-phosphate groups
from DNA, RNA and ribo- and deoxyribonucleoside triphosphates. This enzyme is not
inactivated by heat but can be denatured and removed by phenol extraction. CIAP is
active on 5´ overhangs and 5´ recessed and blunt ends (Sambrook et
al. 1989; Seeburg et al. 1977; Ullrich
et al. 1977; Meyerowitz et al. 1980;
Grosveld et al. 1981).
Additional Resources for Alkaline Phosphatase, Calf Intestinal
Technical Bulletins and Manuals
9PIM182
Alkaline Phosphatase, Calf Intestinal Promega Product
Information
Restriction Enzymes
Restriction enzymes, also referred to as restriction endonucleases, are enzymes
that recognize short, specific (often palindromic) DNA sequences. They cleave
double-stranded DNA (dsDNA) at specific sites within or adjacent to the
recognition sequences. Most restriction enzymes will not cut DNA that is
methylated on one or both strands of their recognition site, although some require
substrate methylation. A complete listing of restriction enzymes available from
Promega can be found on the web.
Additional Resources for Restriction Enzymes
Technical Bulletins and Manuals
TM367
Assembly of Restriction Enzyme Digestions Technical Manual
Promega Publications
eNotes
Rapid DNA digestion using Promega restriction enzymes
eNotes
Activity of Promega restriction enzymes in
GoTaq® Green and PCR Master Mixes
PN081
Work smarter using isoschizomers and neoschizomers
Online Tools
Restriction Enzyme Resource Guide
Citations
Zhang, Y.
et al. (2011) The multidrug efflux pump MdtEF protects against nitrosative damage
during the anaerobic respiration in
Escherichia
coli.
J. Biol. Chem. 286, 26576–84.
The -338 to +39-bp region of tnaC was
amplified from MG1655 genomic DNA using primers incorporating NotI and
HindIII restriction sites at the 5´ and 3´ends of the amplimer,
respectively. After digestion with NotI and HindIII, the PCR product
was gel purified and ligated into a plasmid digested with the same
restriction enzymes so that the lacZ gene in the
plasmid is under control of the tnaC promoter.
Positive clones were confirmed by colony PCR and DNA
sequencing.
PubMed Number:
21642439
Datta, M. and Bhattacharyya, N.P. (2011) Regulation of RE1 protein silencing transcription factor (REST)
expression by HIP1 protein interactor (HIPPI).
J. Biol. Chem. 286, 33759–69.
The upstream promoter region of the mouse REST gene (position
-4773 to -4216) was amplified, digested with BglII and KpnI and cloned
into the same restriction sites of the pGL3 Basic Vector. Five hundred
nanograms of the luciferase reporter construct was transfected into
cells, and after 24 hours, the cells lysed and the luciferase measured
using the Luciferase Reporter Assay.
PubMed Number:
21832040
Transforming a newly constructed plasmid into competent E.
coli cells is the primary method to propagate and select the clone or
clones of interest. Competent bacterial cells are receptive to importing foreign DNA
and replicating it. High-quality competent E. coli is an
integral part of a successful cloning protocol.
JM109 Competent Cells
JM109 Competent Cells (Cat.# L2001) are prepared
according to a modified procedure of Hanahan, 1985. These cells are transformed
with plasmid DNA via the heat-shock method. JM109 cells (Yanisch-Perron
et al. 1985) are an ideal host for many molecular biology
applications and can be used for a-complementation of ß-galactosidase for
blue/white screening.
HB101 Competent Cells
HB101 Competent Cells (Cat.# L2011) are prepared
according to a modified procedure of Hanahan, 1985. HB101 cells (Yanisch-Perron
et al. 1985) are useful for cloning with vectors that do
not require a-complementation for blue/white screening.
Additional Resources for Competent Cells
Technical Bulletins and Manuals
TB095
E. coli Competent Cells Technical Bulletin
Promega Publications
eNotes What are the effects of the bacterial DNA restriction-modification systems on cloning and manipulations of DNA in E. coli?
return to top of page
Materials Required:
(see Composition of Solutions section)
- PCR product (has an A overhang; purification is optional) or blunt DNA
fragment with an A residue added
- pGEM®-T Easy Vector System
(Cat.# A1380) or
pGEM®-T Easy Vector System (Cat.#
A3610)
Both systems include T4 DNA Ligase and chemically competent
high-efficiency JM109 cells.
- Nuclease-Free Water (Cat.# P1193)
-
Optional: 4°C water bath
- LB-Ampicillin plates containing X-Gal and IPTG
- high-efficiency competent cells [e.g., JM109 Competent Cells
(Cat.# L2001)
- SOC medium
- 42°C water bath
- ice
Vector:Insert Ratio
After the insert DNA is prepared for ligation, estimate the concentration by
comparing the staining intensity with that of DNA molecular weight standard of
similar size and known concentrations on an ethidium bromide-stained agarose gel.
If the vector DNA concentration is unknown, estimate the vector concentration by
the same method. Test various vector:insert DNA ratios to determine the optimal
ratio for a particular vector and insert. In most cases, a 1:1 or 1:3 molar ratio
of vector:insert works well. The following example illustrates the calculation of
the amount of insert required at a specific molar ratio of vector:insert.
[(ng of vector × kb size of insert) ÷ kb size of vector] × (molar amount of
insert ÷ molar amount of vector) = ng of insert
Example:
How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a
ligation reaction for a desired vector:insert ratio of 1:3?
[(100ng vector × 0.5kb insert) ÷ 3.0kb vector] × (3 ÷ 1) = 50ng insert
Ligation
- Briefly centrifuge the pGEM®-T or
pGEM®-T Easy Vector and Control Insert DNA
tubes to collect contents at the bottom of the tube.
- Set up ligation reactions as described below. Vortex the 2X Rapid
Ligation Buffer vigorously before each use. Use 0.5ml tubes known to have
low DNA-binding capacity.
|
Reagents |
Standard Reaction |
Positive Control |
Background Control |
2X Rapid Ligation Buffer |
5µl |
5µl |
5µl |
pGEM®-T or
pGEM®-T Easy Vector (50ng) |
1µl |
1µl |
1µl |
PCR product |
Xµl |
– |
– |
Control Insert DNA |
– |
2µl |
– |
T4 DNA Ligase
(3 Weiss units/µl) |
1µl |
1µl |
1µl |
Nuclease-Free Water to a final volume of |
10µl
|
10µl
|
10µl
|
- Mix the reactions by pipetting. Incubate the reactions for 1 hour at room
temperature. Alternatively, incubate the reactions overnight at 4°C for the
maximum number of transformants.
Transformation
- Prepare LB/ampicillin/IPTG/X-Gal plates
(see Composition of Solutions).
- Centrifuge the ligation reactions briefly. Add 2µl of each ligation
reaction to a sterile 1.5 ml microcentrifuge tube on ice. Prepare a
transformation control tube with 0.1ng of an uncut plasmid.
pGEM®-T Vectors are not suitable
for the transformation control as they are linear, not circular.
Note: In our experience, the use of larger (17 × 100mm)
polypropylene tubes (e.g., BD Falcon Cat.# 352059) increases transformation
efficiency. Tubes from some manufacturers bind DNA, thereby decreasing
colony number, and should be avoided.
- Place the high-efficiency JM109 Competent Cells in an ice bath until
just thawed (5 minutes). Mix cells by gently flicking the tube.
- Carefully transfer 50µl of cells to the ligation reaction tubes prepared
in Step 2. Use 100µl of cells for the transformation control tube. Gently
flick the tubes, and incubate on ice for 20 minutes.
- Heat-shock the cells for 45–50 seconds in a water bath at exactly 42°C.
DO NOT SHAKE. Immediately return the tubes to ice for 2 minutes.
- Add 950µl of room temperature SOC medium to the ligation reaction
transformations and 900µl to the transformation control tube. Incubate for
1.5 hours at 37°C with shaking (~150rpm).
- Plate 100µl of each transformation reaction onto duplicate
LB/ampicillin/IPTG/X-Gal plates. For the transformation control, a 1:10
dilution with SOC is recommended prior to plating.
- Incubate plates overnight at 37°C. Select white colonies.
Calculation of Transformation Efficiency
For every transformation with competent cells, we recommend performing a
transformation control using a known quantity of a purified, supercoiled plasmid
DNA (e.g., pGEM®-3Z Vector, Cat.#
P2151). Calculate the transformation efficiency as described
below.
transformation efficiency (cfu/µg) = (cfu on control plate ÷ ng of supercoiled
vector plated) × (103ng/µg) × final dilution factor
cfu = colony forming units
Example:
A 100µl aliquot of competent cells is transformed with 1ng of supercoiled
pGEM®-3Z Vector DNA. Ten microliters of the
transformation reaction (0.1ng total DNA) is added to 990µl of SOC medium (1:100
dilution). Of that volume (1,000µl), a 100µl aliquot is plated (1:1,000 final
dilution), and 100 colonies are obtained on the plate. What is the transformation
efficiency?
(100cfu ÷ 0.1ng of supercoiled vector plated) ×
(103ng/µg) × 1,000 = 1 x 109
cfu/µg
The following protocol is a general procedure to analyze and purify a PCR
fragment. Amplification protocols can be found in the PCR Amplification chapter of
the Protocols and Applications Guide. Additional information
regarding PCR, analysis and product purification can be found in the following
resources:
Amplification
A basic protocol for amplifying genomic DNA by PCR can be found in the PCR Amplification chapter of the Protocols
and Applications Guide, in the section "Example of a PCR
Protocol".
Analysis
Materials Required:
(see Composition of Solutions section)
- aliquot of amplification reaction (usually 5–10µl)
-
Optional: Blue/Orange Loading Dye, 6X (Cat.#
G1881) if GoTaq® Green
Reaction Buffer is not used
- appropriately sized DNA marker
- appropriate agarose gel (typically 0.8–1.2%; see Table 13.1 for
guidelines)
- gel running buffer (1X TAE or 0.5X TBE)
- 10mg/ml ethidium bromide
- Analyze 5–10µl of the amplification reaction using agarose gel
electrophoresis. Include at least one lane containing a DNA size marker to
determine if the PCR products are of the correct size. The products should
be readily visible by UV transillumination of the ethidium bromide-stained
gel (50µg/ml final concentration in the agarose).
- Store reaction products at –20°C until needed.
Table 13.1. Gel Percentages: Resolution of Linear DNA on Agarose Gels. |
Recommended % Agarose |
Optimum Resolution for Linear DNA |
0.5 |
1,000–30,000bp |
0.7 |
800–12,000bp |
1.0 |
500–10,000bp |
1.2 |
400–7,000bp |
1.5 |
200–3,000bp |
2.0 |
50–2,000bp |
If there are primer dimers or at least two PCR products present, the band of
interest will need to be excised and purified (see the next section, PCR Cleanup,
for more information). To minimize the number of extraneous amplimers, the PCR
conditions may need to be optimized. For suggestions on troubleshooting PCR, visit
the PCR Amplification chapter of the Protocols
and Applications Guide
.
PCR Cleanup
Once you have determined that the PCR was successful, you can purify the
desired product from the rest of the reaction components. This can be accomplished
using a number of procedures including direct purification of the product using
the Wizard® SV Gel and PCR Clean-Up System
(Cat.# A9281, A9282, A9285) or separating the
DNA fragments on an agarose gel. Alternatively, you can use a portion of the
amplification reaction directly in a ligation. However, the presence of primer
dimers or other amplimers present can cause false-positive reactions or yield an
incorrect clone (see Figure 13.8). If the reaction is clean (i.e., a single band
is seen on an analytical gel), and the desired product is a minimum of 100bp in
size, you can use the Wizard® SV Gel and PCR Clean-Up
System to directly purify the PCR product [see the DNA
Purification chapter of the Protocols and Applications
Guide
for product protocol].
If there are other bands or a large primer-dimer band present, we recommend gel
electrophoresis to separate the products so that the desired band can be excised.
The DNA can be recovered by melting the excised agarose and using the
Wizard® SV Gel and PCR Clean-Up System.
Optional: A-Tailing Reaction for Blunt-Ended Products:
If a proofreading DNA polymerase was used for amplification and you want to
clone into a T vector, an adenosine residue must be added to the PCR product. This
can be accomplished by incubating the DNA fragment with dATP and a nonproofreading
DNA polymerase, which will add a single 3' A residue. Blunt DNA fragments
resulting from restriction enzyme digestion also can be cloned into T vector after
adding an adenosine residue.
Materials Required:
- blunt-ended product (from PCR or restriction enzyme digestion),
purified
- GoTaq® Flexi DNA Polymerase
- 25mM MgCl2
- 5X GoTaq® Colorless or Green Reaction
Buffer
- 1mM dATP (Cat.# U1205; diluted 1:100 in
nuclease-free water)
- Set up the following reaction in a thin-walled PCR tube:
|
Purified DNA fragment |
1–4.4µl |
5X GoTaq® Reaction Buffer (Colorless
or Green) |
2µl |
1mM dATP (0.2mM final concentration) |
2µl |
GoTaq® Flexi DNA Polymerase (5u/µl) |
1µl |
25mM MgCl2 (1.5mM final concentration) |
0.6µl |
Nuclease-free water to |
10µl
|
- Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.
After the tailing reaction is finished, 1–2µl can be used without further
cleanup for ligation with pGEM®-T or
pGEM®-T Easy Vector Systems.
To determine if the insert was successfully cloned, there are two methods for
screening the transformed bacteria: colony PCR or plasmid miniprep followed by
restriction enzyme digestion.
Successful cloning of an insert into the pGEM®-T and
pGEM®-T Easy Vectors disrupts the coding sequence of
the ß-galactosidase a peptide. Recombinant clones usually can be identified by
blue/white screening on X-Gal/IPTG indicator plates following transformation of
competent cells. However, the characteristics of PCR products cloned into these T
vectors can significantly affect the ratio of blue:white colonies obtained. Clones
that contain PCR products, in most cases, produce white colonies, but blue colonies
can result from PCR fragments that are cloned in-frame with the
lacZ gene. Such fragments are usually a multiple of 3 base pairs
long (including the 3´-A overhangs) and do not contain in-frame stop codons. There
have been reports of DNA fragments of up to 2kb that have been cloned in-frame and
have produced blue colonies.
Even if your PCR product is not a multiple of 3 bases long, the amplification
process can introduce mutations (e.g., deletions or point mutations) that may result
in blue colonies when competent cells are transformed with the fragment inserted into
the pGEM®-T or pGEM®-T Easy
Vectors.
Screening of recombinant clones using restriction enzymes is more time-consuming
than colony PCR and involves isolating the plasmid DNA from liquid cultures of
individual E. coli colonies, performing the restriction enzyme
digestion and determining if the insert is of the correct size. To learn more about
screening by restriction enzyme digestion, visit the Subcloning
Notebook.
The following protocol is for colony PCR analysis of transformants.
Materials Required:
(see Composition of Solutions section)
- plate of colonies containing the recombinant plasmid
- toothpicks or sterile bacterial loop
- LB Broth (optional)
- upstream screening primer
- downstream screening primer
- GoTaq® Flexi DNA Polymerase
- 5X Green GoTaq® Flexi Buffer
- 25mM MgCl2
- Nuclease-Free Water (Cat.# P1193)
- Nuclease-Free Light Mineral Oil (e.g., Sigma Cat.# M5904 or Promega
Cat.# DY1151) if you are using a thermal
cycler without a heated lid; do not autoclave
- dNTP Mix (10mM of each dNTP; Cat.# U1511,
U1515)
- Pick a well-isolated colony using either a sterile toothpick or a flamed and
cooled bacterial loop, and transfer to 50µl of sterile water. Part of the
colony may be transferred to LB medium containing the appropriate antibiotic
for overnight culture and plasmid miniprep, if desired.
- Boil for 10 minutes to break open the bacterial cell wall and release the
DNA.
- Centrifuge at 16,000 × g for 5 minutes to pellet the
cell debris.
- Use 5µl of the supernatant in a 50µl amplification reaction (see Table 13.2
for a sample reaction).
Table 13.2. Colony PCR using GoTaq® Flexi DNA Polymerase. |
Components |
Volume |
Final Concentration |
Nuclease-Free Water (to a final volume of 50µl) |
Xµl |
|
5X Reaction Buffer |
10µl |
1X |
dNTP mix (10mM of each dNTP) |
1µl |
0.2mM each |
GoTaq® DNA polymerase (5u/µl) |
0.25µl |
0.025u/µl |
25mM MgCl2
|
3µl |
1.5mM |
Downstream screening primer |
50pmol1
|
1µM |
Upstream screening primer |
50pmol1
|
1µM |
Boiled colony supernatant |
5µl |
|
1A general formula for calculating the number of nanograms of primer
equivalent to 50pmol is: 50pmol = 16.3ng × b; where b is the number of
bases in the primer.
- Amplify the target DNA using cycling conditions appropriate for your
screening primers and size of amplimer (see Table 13.3 for suggestions). Place
reactions in a thermal cycler that has been preheated to 94°C.
Table 13.3. Suggested Amplification Conditions. |
Step |
Temperature |
Time (minutes) |
Cycles |
Initial denaturation |
94°C |
2 |
1 |
Denaturation |
94°C |
0.5–1.0 |
25–35 |
Annealing |
42–65°C1
|
0.5–1.0 |
|
Extension |
72°C |
1 minute/
kilobase2
|
|
Final extension |
72°C |
5 |
1 |
Soak/Hold |
4°C |
Indefinite |
1 |
1Annealing temperature should be optimized for each primer set based on
the primer melting temperature (Tm). To calculate
melting temperatures of primers in GoTaq®
Reaction Buffer, go to BioMath
Calculators.
2The extension time should be at least 1 minute per kilobase of target.
Typically, amplimers smaller than 1kb use a 1-minute extension.
- Remove an aliquot of the completed PCR and analyze by agarose gel
electrophoresis for the product of appropriate size, which indicates the
correct insert is present in the clone.
-
Recommended: Culture the appropriate colony or colonies to create
a glycerol stock of your recombinant plasmid or plasmids, and purify the
plasmids in larger quantities [e.g., PureYield™ Plasmid Systems
(Cat.# A2492, A2495)] for downstream
applications or further manipulation.
Classic subcloning involves restriction digestion of the plasmid of interest to
remove the desired DNA fragment followed by ligation into a second vector with
compatible ends. PCR can be used for subcloning as well, amplifying the insert from
one plasmid and cloning the product into a T vector. Alternatively, the PCR product
can be generated using primers with restriction enzyme sites, cut with the
appropriate enzymes, then cloned into a vector with compatible ends. Further
information on subcloning can be found by visiting the Subcloning
Notebook.
return to top of page
The desired protein-coding region must be amplified by PCR before being cloned
into the Flexi® Vectors
(Figures 13.6 and 13.7). The optimal conditions for amplifying the
protein-coding region will depend on the DNA template, DNA polymerase, PCR primers
and other reaction parameters. We recommend following the protocol provided with the
DNA polymerase to generate the PCR product. For protein-coding regions less than
700bp, consider using GoTaq® DNA Polymerase to amplify
your protein-coding region. For regions greater than 700bp, we recommend the use of a
high-fidelity DNA polymerase, such as Pfu DNA polymerase. To
facilitate cloning, the PCR primers used to amplify the protein-coding region must
append an SgfI site and a PmeI site to the PCR product. To append these sites,
incorporate an SgfI site in your amino-terminal PCR primer and a PmeI site in your
carboxy-terminal PCR primer. Transfer of protein-coding regions into N-terminal
Flexi® Vectors results in translational readthrough of
the SgfI site, which encodes the peptide sequence Ala-Ile-Ala. The PmeI site is
placed at the carboxy terminus, appending a single valine residue to the last amino
acid of the protein-coding region. The valine codon, GTT, is immediately followed by
an ochre stop codon, TAA. Primer design guidelines are provided in Technical Manual #TM254 and the Flexi® Vector Primer Design Tool.
To cleanup the PCR product, refer to Amplification,
Analysis and PCR Cleanup.
Digestion reactions for the PCR product and the acceptor
Flexi® Vector can be performed concurrently.
Note: Do not use C-terminal Flexi® Vectors,
which have names starting with ”pFC”, as acceptors for PCR products if you plan to
transfer the protein-coding region to a different Flexi®
Vector in the future. C-terminal Flexi® Vectors lack PmeI
sites and cannot serve as donors for other Flexi® Vectors.
Materials Required:
- Flexi® System, Entry/Transfer
(Cat.# C8640)
- chosen acceptor Flexi® Vector
- purified PCR product
- Thaw the 5X Flexi® Digest Buffer, the acceptor
Flexi® Vector and Nuclease- Free Water, and store
on ice. Vortex the 5X Flexi® Digest Buffer and the
acceptor Flexi® Vector before use.
- Combine the following reaction components to cut the PCR product with SgfI
and PmeI.
|
Component |
Volume |
5X Flexi® Digest Buffer |
4µl |
Purified PCR product (up to 500ng) |
Xµl |
Flexi® Enzyme Blend (SgfI and PmeI) |
4µl |
Nuclease-Free Water to a final volume of |
20µl
|
- Combine the following reaction components to cut the acceptor
Flexi® Vector with SgfI and PmeI.
|
Component |
Volume |
Nuclease-Free Water |
12µl |
5X Flexi® Digest Buffer |
4µl |
Acceptor Flexi® Vector (200ng) |
2µl |
Flexi® Enzyme Blend (SgfI and PmeI) |
2µl |
Final Volume of |
20µl
|
Note: Take care when pipetting solutions that contain glycerol, such
as the Flexi® Enzyme Blend, because small volumes are
difficult to pipet accurately.
- Incubate both reactions (Steps 2 and 3) at 37°C for
30 minutes.
- Heat the reaction with the Flexi® Vector (Step 3)
at 65°C for 20 minutes to inactivate the restriction enzymes. Store on ice
until the PCR product and vector are ligated in Ligation
of PCR Product and Acceptor Flexi®
Vector.
- Directly purify the digested PCR product using the
Wizard® SV Gel and PCR Cleanup System
(Cat.# A9281).
- Combine the following reaction components:
- Incubate at room temperature for 1 hour.
Note: The 2X Flexi® Ligase Buffer
contains ATP, which degrades during temperature fluctuations. Avoid multiple
freeze-thaw cycles and exposure to frequent temperature changes by making
single-use aliquots of the buffer.
Note: Do not use this protocol to screen for inserts in C-terminal
Flexi® Vectors, which have names starting with “pFC”,
since these clones lack PmeI sites.
Materials Required:
(see Composition of Solutions section)
- Place reaction components and reaction tubes or plates on ice.
- Prepare a master mix by combining the components listed below. Increase
volumes proportionately depending on the number of reactions.
|
Component |
Volume per Reaction |
Nuclease-Free Water |
10.5µl |
5X Flexi® Digest Buffer |
4µl |
10X Flexi® Enzyme Blend (SgfI and PmeI) |
2µl |
Final Volume of |
15µl
|
- Add 15µl of master mix to 5µl (200–500ng) of plasmid DNA. Mix thoroughly by
pipetting.
- Incubate for 2 hours at 37°C.
- Add 5µl of loading dye (Blue/Orange Loading Dye, 6X, Cat.#
G1881). Incubate at 65°C for 10 minutes.
- Load 20µl of the reaction onto a 1% agarose gel and separate fragments by
electrophoresis. Visualize the fragments by ethidium bromide staining.
Transfer refers to moving your protein-coding region from one
Flexi® Vector (donor) to another
Flexi® Vector (acceptor). Choose an appropriate acceptor
vector with the desired expression and tag options and a different antibiotic
resistance marker than the donor because antibiotic selection is the basis for
selecting the desired clone
(Figures 13.5, 13.9 and 13.10).
There are two basic categories of Flexi® Vectors: those
containing SgfI and PmeI sites and expressing either a native (untagged) protein or
an N-terminal-tagged protein, and those containing SgfI and EcoICRI sites and
expressing a C-terminal-tagged protein. Flexi® Vectors for
expressing C-terminal-tagged proteins act only as acceptors, never as donor vectors.
To transfer protein-coding regions between Flexi® Vectors
expressing native protein or an N-terminal-tagged protein, the donor and acceptor
vectors are digested with SgfI and PmeI simultaneously, prior to ligation of the
insert, transformation and selection of the cells (Figure 13.9).
To create a C-terminal-tagged protein, the donor plasmid expressing native protein
or an N-terminal-tagged protein is digested with SgfI and PmeI. Because EcoICRI cuts
frequently in protein-coding regions, the acceptor plasmid containing the C-terminal
tag is digested with SgfI and EcoICRI in a separate reaction. The two separate
digests are combined for ligation of the insert, transformation and selection of the
cells (Figure 13.10).
Transfer of Protein-Coding Regions Between Flexi®
Vectors Expressing Native or N-Terminal Fusion Proteins
Materials Required:
- Flexi® System, Transfer
(Cat.# C8820)
- competent E. coli cells [e.g., JM109 Competent
Cells (Cat.# L2001)]
- LB plates supplemented with the appropriate antibiotic at the
appropriate concentration (see Composition of
Solutions and Figure 13.5)
- Use the Wizard®
Plus SV Minipreps DNA Purification System
(Cat.# A1330),
Wizard® SV 96 Plasmid DNA Purification System
(Cat.# A2250) or a similar method to
prepare the donor Flexi® Vector DNA [see the
DNA Purification chapter of the
Protocols and Applications Guide
]. Adjust the volume so that the final DNA concentration is
50–100ng/µl.
- Combine the following reaction components to cut the
Flexi® Vectors:
|
Component |
Volume |
5X Flexi® Digest Buffer |
4µl |
Acceptor Flexi® Vector (100ng) |
1µl |
Donor Flexi® Vector (100ng) |
Xµl |
Flexi® Enzyme Blend (SgfI and PmeI) |
2µl |
Nuclease-Free Water to a final volume of |
20µl
|
Note: Take care when pipetting solutions that contain glycerol, such as the
Flexi® Enzyme Blend, because small volumes are
difficult to pipet accurately.
- Incubate at 37°C for 15–30 minutes.
- Heat the reaction at 65°C for 20 minutes to inactivate the restriction
enzymes. Store the reaction on ice while assembling the ligation reaction in
Step 5.
- Combine the following ligation reaction components:
|
Component |
Volume |
2X Flexi® Ligase Buffer |
10µl |
Digested DNA from Step 4 (100ng total) |
10µl |
T4 DNA Ligase (HC; 20u/µl) |
1µl |
Final volume of |
21µl
|
- Incubate at room temperature for 1 hour.
- Transform the ligation reaction into high-efficiency,
E. coli competent cells (=1 ×
108cfu/µg DNA). If you are using competent cells
other than high-efficiency JM109 Competent Cells (Cat.#
L2001) purchased from Promega, it is important to follow the
appropriate transformation protocol. The recommended transformation protocol
for our high-efficiency JM109 Competent Cells is provided in Ligation and Transformation. Selection for
transformants should be on LB plates supplemented with 100µg/ml ampicillin
for Flexi® Vectors with the letter “A” in the
name or 30µg/ml kanamycin for Flexi® Vectors with
the letter “K” in the name. See Figure 13.5 for a list of
antibiotic-resistance genes carried on the various vectors.
- Screen at least four colonies for each protein-coding region. Digest the
plasmid to ensure that SgfI and PmeI cleave their recognition sites flanking
the protein-coding region so that the insert can be cloned into other
Flexi® Vectors.
Screen at least eight colonies for each protein-coding region transferred
to the pF3A WG (BYDV) or pF3K WG (BYDV) Flexi®
Vectors. Lower transfer frequencies with these vectors are due to a higher
background of plasmid backbone heterodimers between the WG (BYDV) Vectors
and other Flexi® Vectors. Other
Flexi® Vectors share common regions flanking
the SgfI and PmeI sites such that plasmid backbone dimers are unstable
(Yoshimura et al. 1986). The pF3A and pF3K WG
(BYDV) Flexi® Vectors lack these common flanking
regions due to the inclusion of the BYDV translation-enhancing sequences.
If you are using the pF3A WG (BYDV) or pF3K WG (BYDV)
Flexi® Vectors, the number of minipreps performed
can be reduced by prescreening colonies to identify those harboring plasmid
backbone heterodimers. Colonies containing such heterodimers can be
identified by their ability to grow on both antibiotics. Pick individual
colonies, and restreak on an ampicillin plate and a kanamycin plate or
inoculate medium containing ampicillin and medium containing kanamycin. Grow
overnight. Colonies containing the clone of interest will grow only in the
antibiotic associated with the acceptor plasmid.
Transfer of Protein-Coding Regions from Native or N-Terminal
Flexi® Vectors to C-Terminal
Flexi® Vectors
Use the C-terminal Flexi® Vectors, which have names
starting with "pFC", as acceptors but not as donors because they lack PmeI sites.
Materials Required:
- Carboxy Flexi® System, Transfer
(Cat.# C9320)
- competent E. coli cells [e.g., JM109 Competent
Cells (Cat.# L2001)]
- LB plates supplemented with the appropriate antibiotic at the
appropriate concentration (see Composition of
Solutions and Figure 13.5)
- Use the Wizard®
Plus SV Minipreps DNA Purification System
(Cat.# A1330),
Wizard® SV 96 Plasmid DNA Purification System
(Cat.# A2250) or a similar method to
prepare the donor Flexi® Vector DNA [see the
DNA Purification chapter of the
Protocols and Applications Guide
]. Adjust the volume so that the final DNA concentration is
50–100ng/µl.
- Combine the following reaction components to cut the donor
Flexi® Vector:
|
Component |
Volume |
5X Flexi® Digest Buffer |
2µl |
Donor Flexi® Vector (100ng) |
Xµl |
Flexi® Enzyme Blend (SgfI and PmeI) |
1µl |
Nuclease-Free Water to a final volume of |
10µl
|
Note: Take care when pipetting solutions that contain glycerol, such as the
Flexi® Enzyme Blend, because small volumes are
difficult to pipet accurately.
- In a separate tube, combine the following reaction components to cut the
acceptor C-terminal Flexi® Vector:
|
Component |
Volume |
Nuclease-Free Water |
6µl |
5X Flexi® Digest Buffer |
2µl |
Acceptor C-Terminal Flexi® Vector
(100ng)1
|
1µl |
Carboxy Flexi® Enzyme Blend (SgfI
and EcoICRI) |
1µl |
Final volume |
10µl
|
1Acceptor C-terminal Flexi® Vectors will
have names starting with "pFC".
- Incubate both reactions at 37°C for 15–30 minutes.
- Heat both reactions at 65°C for 20 minutes to inactivate the restriction
enzymes. Store the reactions on ice while assembling the ligation reaction
in Step 6.
- Combine the following ligation reaction components:
|
Component |
Volume |
2X Flexi® Ligase Buffer |
10µl |
Digested donor Flexi® Vector
prepared in Step 2 (approximately 50ng) |
5µl |
Digested acceptor C-terminal Flexi®
Vector prepared in Step 3 (50ng) |
5µl |
T4 DNA Ligase (HC; 20u/µl) |
1µl |
Nuclease-Free Water to a final volume of |
21µl
|
- Incubate at room temperature for 1 hour.
- Transform the ligation reaction into high-efficiency,
E. coli competent cells (=1 ×
108cfu/µg DNA). If you are using competent cells
other than high-efficiency JM109 Competent Cells (Cat.#
L2001) purchased from Promega, it is important to follow the
appropriate transformation protocol. The recommended transformation protocol
for our high-efficiency JM109 Competent Cells is provided in Ligation and Transformation. Selection for
transformants should be on LB plates supplemented with 100µg/ml ampicillin
for Flexi® Vectors with the letter “A” in the
name or 30µg/ml kanamycin for Flexi® Vectors with
the letter “K” in the name. See Figure 13.5 for a list of
antibiotic-resistance genes carried on the various vectors.
- Screen at least eight colonies for each protein-coding region. Successful
plasmid constructs will not cut with PmeI but will cut with SgfI. Lower
transfer frequencies are due to a higher background of plasmid backbone
heterodimers between the C-terminal Flexi®
Vectors and other Flexi® Vectors. Other
Flexi® Vectors share common regions flanking
the SgfI and PmeI sites such that plasmid backbone dimers are unstable
(Yoshimura et al. 1986). The C-terminal
Flexi® Vectors may lack these common flanking
regions due to the inclusion of the protein fusion tag sequence.
return to top of page
Materials Required:
- Nuclease-Free Water (Cat.# P1193)
- DNA Polymerase I Large (Klenow) Fragment and 10X Reaction Buffer
(Cat.# M2201) or T4 DNA Polymerase
(Cat.# M4211)
- T4 DNA Polymerase 10X buffer (optional)
- Bovine Serum Albumin (BSA), Acetylated, 1mg/ml (Cat.#
R9461)
- dNTPs, 100mM (Cat.# U1240)
Both Klenow (DNA Polymerase I Large Fragment) and T4 DNA Polymerase can be used
to fill 5'-protruding ends with deoxynucleotide triphosphates (dNTPs). Properties
of these enzymes are discussed in Anderson et al. 1980 and
Challberg and Englund, 1980.
Klenow Fragment Method
For optimal activity, use the Klenow 10X Buffer supplied with the enzyme. DNA
Polymerase I Large (Klenow) Fragment is also active in many restriction enzyme
buffers, and some users may choose to perform the fill-in reaction directly in the
restriction buffer.
- Optional: Following the restriction enzyme digestion that generated the
5'-protruding ends, purify the DNA using the
Wizard® SV Gel and PCR Clean-Up System
(Cat.# A9281) or other DNA purification
system. Alternatively, the DNA can be extracted with phenol:chloroform,
ethanol precipitated and rehydrated for use in the conversion
reaction.
- Proceed with one of the following for the fill-in reaction (total
reaction volume can be 10–100µl.):
For DNA purified over a column: The optimal reaction conditions
for filling in are: 1X Klenow Reaction Buffer [50mM Tris- HCl (pH 7.2), 10mM
MgSO4, 0.1mM DTT], 40µM of each dNTP, 20µg/ml
acetylated BSA and 1 unit of Klenow Fragment per microgram of DNA.
For digested DNA in restriction enzyme buffer: Klenow Fragment
is also partially active in many restriction enzyme buffers (such as our
4-CORE® Buffers), and the fill-in reaction may
be performed directly in the restriction enzyme buffer supplemented with
40µM of each dNTP, thereby eliminating the clean-up step. Add 1 unit of
Klenow Fragment per microgram of DNA.
For ethanol-precipitated DNA: Resuspend DNA in Klenow 1X Buffer
containing 40µM of each dNTP and 20µg/ml of Acetylated Bovine Serum Albumin
(BSA). Add 1 unit of Klenow Fragment per microgram of DNA.
- Incubate the reaction at room temperature for
10 minutes.
- Stop the reaction by heating at 75°C for 10 minutes.
T4 DNA Polymerase Method
Prepare the DNA as described for the Klenow Fragment method. Like the Klenow
Fragment, T4 DNA Polymerase functions well in many restriction enzyme buffers. Add
5 units of T4 DNA Polymerase per microgram of DNA, 100µM of each dNTP and
0.1mg/ml Acetylated BSA. The recommended reaction buffer for T4 DNA Polymerase is
1X T4 DNA Polymerase Buffer. Incubate the reaction at 37°C for 5 minutes. Stop the
reaction by heating at 75°C for 10 minutes or by adding 2µl of 0.5M EDTA.
Materials Required:
Note: T4 DNA Polymerase has a 3'?5' exonuclease
activity that will, in the presence of excess dNTPs, convert a 3'-protruding end
to a blunt end (Burd and Wells, 1974).
- Following the restriction enzyme digestion that generates 3'-protruding
ends, leave the DNA in restriction enzyme buffer, exchange the buffer for 1X T4
DNA Polymerase Buffer or gel purify the desired fragment (see the PCR Cleanup
section of Amplification, Analysis and PCR
Cleanup).
- Add 5 units of T4 DNA Polymerase per microgram of DNA and 100µM of each
dNTP.
- Incubate at 37°C for 5 minutes.
Note: At high concentrations of dNTPs (100µM), degradation of the
DNA will stop at duplex DNA; however, if the dNTP supply is exhausted, the very
active exonuclease activity (200 times more active than that of DNA polymerase
I) will degrade the double-stranded DNA (Sambrook et al.
1989).
- Stop the reaction by heating at 75°C for 10 minutes or adding 2µl of 0.5M
EDTA.
If the ends of the prepared vector are identical (e.g., following a single
digestion), it is advantageous to treat the vector with TSAP Thermosensitive Alkaline
Phosphatase (Cat.# M9910) to remove the phosphate
groups from the 5' ends to prevent self-ligation of the vector (Sambrook et
al. 1989). For linear vectors with unique 5' ends, TSAP treatment is
not necessary.
Note: Since TSAP is active in all Promega restriction enzyme buffers,
the vector DNA easily can be restriction digested and dephosphorylated at the same
time. The following protocol reflects this streamlined method. See the
TSAP Thermosensitive Alkaline Phosphatase Product Information
#9PIM991 for alternative protocols.
- As a general guideline, for reactions containing up to 1µg of DNA, add 15
units of restriction enzyme and the amount of TSAP listed below to the vector
DNA in a total reaction volume of 20–50µl. Set up the reaction in the
appropriate 1X Promega restriction enzyme reaction buffer.
|
Reaction Buffer |
Amount of TSAP for Reactions Containing =1µg DNA |
Promega 10X Reaction Buffers A–L (except F) |
1µl |
Promega 10X Reaction Buffer F |
2µl |
MULTI-CORE™ 10X Buffer |
1µl |
- Incubate the reaction at 37°C for 15 minutes. This is sufficient to digest
and dephosphorylate all vector DNA overhang types (3´, 5´ or blunt).
- Heat-inactivate the TSAP and restriction enzyme by incubating the reaction
at 74°C for 15 minutes.
Note: Not all restriction enzymes can be heat-inactivated. If the
restriction enzyme cannot be heat-inactivated, clean up the digest using the
Wizard® SV Gel and PCR Clean-Up System
(Cat.# A9281).
- Briefly centrifuge the reaction, and use approximately 40ng of
dephosphorylated vector in a ligation reaction containing DNA insert, 1X Rapid
Ligation Buffer and 2µl (6 units) of T4 DNA Ligase (LigaFast™ Rapid DNA
Ligation System, Cat.# M8221). Incubate ligation
reactions containing vector with 5´ or 3´ overhangs at 25°C for 5 minutes.
Incubate ligation reactions containing vector with blunt ends at 25°C for
15 minutes.
Note: Optimal vector:insert ratios may need to be determined. We
recommend using a 1:2 molar ratio of vector to insert DNA as a starting point.
See the LigaFast™ Rapid DNA Ligation System Product Information
#9PIM822 for additional information.
- Transform the ligated material directly into E. coli
competent cells following the recommended transformation protocol provided with
the cells.
return to top of page
antibiotic stock solutions
100mg/ml
ampicillin in deionized water (sterile filtered)
25mg/ml
kanamycin; kanamycin sulfate in deionized water (sterile
filtered)
Store at –20°C.
Blue/Orange 6X Loading Dye
5X Flexi® Digest Buffer
50mM
Tris-HCl (pH 7.9 at 37°C)
2X Flexi® Ligase Buffer
60mM
Tris-HCl (pH 7.8 at 25°C)
Store in single-use aliquots at –20°C. Avoid multiple freeze-thaw
cycles.
IPTG stock solution (0.1M)
1.2g
isopropyl ß-D-thiogalactopyranoside
Add deionized water to 50ml final volume. Filter sterilize, and store at
4°C.
LB medium
Add deionized water to approximately 1L. Adjust pH to 7.5 with 10N NaOH, and
autoclave. For LB plates, include 15g agar prior to autoclaving.
LB plates with antibiotic
Add 15g agar to 1 liter of LB medium. Autoclave. Allow the medium to cool to
50°C before adding ampicillin to a final concentration of 100µg/ml or kanamycin to
a final concentration of 30µg/ml, as appropriate for the acceptor
Flexi® Vector. Pour 30–35ml of medium into 85mm
petri dishes. Let the agar harden. Store at 4°C for up to 1 month or at room
temperature for up to 1 week.
LB plates with ampicillin/IPTG/X-Gal
Make the LB plates with ampicillin as described above, then supplement with
0.5mM IPTG and 80µg/ml X-Gal and pour the plates. Alternatively, spread 100µl of
100mM IPTG and 20µl of 50mg/ml X-Gal over the surface of an LB-ampicillin plate
and allow to absorb for 30 minutes at 37°C prior to use.
2M Mg2+ stock
Add distilled water to 100ml. Filter sterilize.
SOC medium (100ml)
0.5g
Bacto®-yeast extract
1ml
2M Mg2+ stock, filter-sterilized
1ml
2M glucose, filter-sterilized
Add Bacto®-tryptone,
Bacto®-yeast extract, NaCl and KCl to 97ml of distilled
water. Stir to dissolve. Autoclave and cool to room temperature. Add 2M
Mg2+ stock and 2M glucose, each to a final
concentration of 20mM. Bring the volume to 100ml with sterile, distilled water.
The final pH should be 7.0.
X-Gal (2ml)
100mg
5-bromo-4-chloro-3-indolyl-ß-D-galactoside
Dissolve in 2ml N,N´-dimethyl-formamide. Cover with aluminum foil, and store at
–20°C. Alternatively, use 50mg/ml X-Gal (Cat.#
V3941).
return to top of page
- Anderson, S. et al.
(1980) A short primer for sequencing DNA cloned in the single-stranded phage vector
M13mp2.
Nucleic Acids Res.
8, 1731–43.
- Burd, J.F. and Wells, R.D. (1974) Synthesis and characterization of the duplex block polymer
d(C15A15)-d(T15G15).
J. Biol. Chem.
249, 7094–101.
- Challberg, M.D. and Englund, P.T. (1980) Specific labeling of 3' termini with T4 DNA polymerase.
Methods Enzymol.
65, 39–43.
- Clark, J.M. (1988) Novel non-templated nucleotide addition reactions catalyzed by procaryotic and
eucaryotic DNA polymerases.
Nucleic Acids Res.
16, 9677–86.
- D’Avino, P.P. et al.
(2004) Mutations in sticky lead to defective organization of the
contractile ring during cytokinesis and are enhanced by Rho
and suppressed by Rac.
J. Cell Biol.
166, 61–71.
- Engler, M.J. and Richardson, C.C. (1982) In: The Enzymes,Boyer, P.D., ed., Academic Press, New York, NY.
- Grosveld, F.G. et al.
(1981) Isolation of ß-globin-related genes from a human cosmid library.
Gene
13, 227–37.
- Hanahan, D. (1985)
DNA Cloning, Vol. 1, Glover, D., ed., IRL Press, Ltd.,
109.
- Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA polymerase-generated PCR
fragments into pGEM®-T Vector Systems.
Promega Notes
71, 10–4.
- Le Gall, S.M. et al.
(2003) Regulated cell surface pro-EGF ectodomain shedding is a zinc
metalloprotease-dependent process.
J. Biol. Chem.
278, 45255–68.
- Meyerowitz, E.M. et al.
(1980) A new high-capacity cosmid vector and its use.
Gene
11, 271–82.
- Mezei, L.M. and Storts, D.R. (1994) Purification of PCR products. In: PCR Technology: Current
Innovations,
Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL,
21.
- Newton, C.R. and Graham, A. (1994) In: PCR,BIOS Scientific Publishers, Ltd., Oxford, UK, 13.
- Robles, J. and Doers, M. (1994) pGEM®-T Vector Systems troubleshooting guide.
Promega Notes
45, 19–20.
- Sakakida, Y. et al.
(2005) Importin a/ß mediates nuclear transport of a mammalian circadian clock
component, mCRY2, together with mPER2, through a bipartite nuclear localization
signal.
J. Biol. Chem.
280, 13272–8.
- Sambrook, J. et al.
(1989)
Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.
- Seeburg, P.H. et al.
(1977) Nucleotide sequence and amplification in bacteria of structural gene for rat
growth hormone.
Nature
270, 486–94.
- Studier, F.W. and Moffat, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level
expression of cloned genes.
J. Mol. Biol.
189, 113–30.
- Ullrich, A. et al.
(1977) Rat insulin genes: Construction of plasmids containing the coding sequences.
Science
196, 1313–9.
- Yanisch-Perron, C. et al.
(1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of
the M13mp18 and pUC19 vectors.
Gene
33, 103–19.
- Yoshimura, H. et al.
(1986) Biological characteristics of palindromic DNA (II).
J. Gen. Appl. Microbiol.
32, 393–404.
return to top of page
4-CORE, Flexi, GoTaq, HaloTag, pGEM and Wizard are registered trademarks of
Promega Corporation.
LigaFast, MULTI-CORE, PureYield and
pTARGET are trademarks of Promega Corporation.
Bacto is a registered trademark of Difco Laboratories, Detroit, Michigan.
Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. DH5a is a
trademark of Life Technologies, Inc. Ficoll is a registered trademark of GE
Healthcare Bio-sciences. Gateway is a registered trademark of Invitrogen Corporation.
SYBR is a registered trademark of Molecular Probes, Inc.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our web
site for more information.
All prices and specifications are subject to change without
prior notice.
Product claims are subject to change. Please contact
Promega Technical Services or access the Promega online catalog for the
most up-to-date information on Promega products.