Chapter 13: Cloning

Introduction

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 Protocols and Applications Guide chapter on PCR Applications.

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 the 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 religating (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 α-complementation to regenerate
β-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,
β-galactosidase, is produced when the lacZ coding information missing on the F´ episome is provided by the information contained in the plasmid. This activity is detected by plating bacteria transformed by plasmids on plates containing isopropyl β-D-thiogalactopyranoside (IPTG; an inducer of the lac promoter) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal; a dye that produces a blue color when hydrolyzed by β-galactosidase). When the reading frame of the α peptide is disrupted by insertion of a foreign DNA fragment or deletion of vector sequences, α-complementation does not occur, and the bacterial colonies remain white or occasionally light blue.

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Promega Products for Cloning

Thermostable DNA Polymerases

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 these 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 listing of the various enzymes for use in PCR can be found in the Protocol and Applications Guide chapter on PCR Applications, 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 three different formulations to suit your needs: the standard GoTaq^® DNA Polymerase, which is supplied with 1.5mM MgCl₂ in the 1X reaction buffer; GoTaq^® Flexi DNA Polymerase, which allows a range of MgCl₂ to be added for PCR; and GoTaq^® Green Master Mix, which is a premixed, ready-to-use solution containing GoTaq^® DNA Polymerase, dNTPs, MgCl₂ and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR. All GoTaq^® products contain Taq DNA polymerase in a proprietary formulation that offer 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.

T-Cloning Vectors

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 the cloning of PCR products. The vectors are prepared by cutting with a blunt-ended restriction endonuclease 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 the efficiency of ligation of a PCR product into the plasmids 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 α-peptide of β-galactosidase. Insertional inactivation of the α-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 the 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 in which direction the insert was cloned. To verify the direction of the insert, amplify recombinant plasmids using one of the gene specific PCR primers and each of the phage promoter primers, which are present on the pGEM^®-T Vector (Knoche and Kephart, 1999). The correct orientation is important for transcription or translation or both.

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 α peptide of β-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).

Flexi^® Vector Systems

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 by primers designed with two rare-cutting restriction enzymes, SgfI and PmeI. Then, after restriction enzyme digestion, the insert can ligate 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 the 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 system overview and Figure 13.5 for list of example vectors). A current list of available vectors can be found at: www.promega.com/catalog/catalogredirect.aspx?partno=c8640. 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 the 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 act as acceptors of 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).

Modifying and Restriction Enzymes

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 a PCR product, or other type of insert, to generate compatible ends for ligation. REs can also evaluate the success of the ligation 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 cohesive-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 also 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).

The LigaFast™ Rapid DNA Ligation System (Cat.# M8221, M8225) is designed for the 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 is designed to eliminate any further purification prior to transformation of ligated DNA. The specially formulated 2X Rapid Ligation Buffer requires no additional ATP or Mg²⁺ prior to use.

Alkaline Phosphatases

Alkaline phosphatases catalyze the 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 may also 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 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 is also effectively and irreversibly inactivated by heating at 74°C for
15 minutes. Therefore, a DNA cleanup step is not required before ligation.

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).

Shrimp Alkaline Phosphatase (SAP; Cat.# M8201) catalyzes the dephosphorylation of 5´ phosphates from DNA and RNA. Unlike Calf Intestinal Alkaline Phosphatase, SAP is completely and irreversibly inactivated by heating at 65°C for 15 minutes. SAP is active on 5´ overhangs and 5´ recessed and blunt ends (Sambrook et al. 1989).

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 their recognition sequences. Most restriction enzymes (REs) 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.

Competent Cells

Transforming a newly constructed plasmid into competent E. coli cells is the primary method to propagate and select for 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 α-complementation of β-galactosidase for blue/white screening.

Single Step (KRX) Competent Cells

Single Step (KRX) Competent Cells (Cat.# L3001, L3002) are not only highly competent and compatible with blue/white screening but can be used for tightly controlled protein expression. KRX incorporates a chromosomal copy of the T7 RNA polymerase gene driven by a rhamnose promoter (rhaBAD). T7 RNA polymerase-based systems (Studier and Moffat, 1986) are one of the most widely used protein expression systems by virtue of its well-defined promoter, which is completely independent of E. coli RNA polymerase promoters, and its rapid elongation rate, about five times that of E. coli RNA polymerases. The rhaBAD promoter is subject to catabolite repression by glucose, is activated by addition of rhamnose to the medium, and provides precise control of T7 RNA polymerase abundance and thereby precise control of recombinant protein production.

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 α-complementation for blue/white screening.

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PCR Cloning Protocols

Amplification, Analysis and PCR Cleanup

The following protocol is a general procedure to analyze and purify a PCR fragment. Protocols for amplification can be found in the Protocols and Applications Guide chapter on PCR Applications. Additional information regarding PCR, analysis and product purification can be found in the following resources:

• Protocols and Applications Guide chapter on PCR Applications
• Protocols and Applications Guide chapter on DNA Purification

Amplification

A basic protocol for amplifying genomic DNA by PCR can be found in the Protocols and Applications Guide chapter on PCR Applications, 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 percentage 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

1. 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).

2. Store reaction products at –20°C until needed.

If there are primer dimers or at least two PCR products present, the band of interest will need to be removed 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 Protocols and Applications Guide chapter on PCR Applications.

PCR Cleanup

Once you have determined 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 the Wizard^® SV Gel and PCR Clean-Up System (Cat.# A9280, A9281, A9282) or agarose gel isolation. 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 seen on an analytical gel) and 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 Protocols and Applications Guide chapter on DNA Purification for product protocol].

If there are other bands or a large primer-dimer band present, we recommend gel electrophoresis to separate the products so the desired band can be excised. The DNA can be recovered using an agarose-digesting enzyme such as AgarACE™ Enzyme (Cat.# M1741, M1743) or 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 onto 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 can also 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)

1. Set up the following reaction in a thin-walled PCR tube:

2. 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.

Ligation and Transformation

Materials Required:
(see Composition of Solutions section)

• PCR product (has an A overhang and optional purification) 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 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) or Single Step KRX Competent Cells (Cat.# L3001)]
• SOC medium
• 42°C water bath
• ice

Vector:Insert Ratio

After the insert DNA has been prepared for ligation, estimate the concentration by comparing the staining intensity with that of DNA molecular weight standards of 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, either a 1:1 or a 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.

[ng of vector × size of insert (in kb)] ÷ size of vector (in kb) × molar amount of (insert ÷ 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

1. Briefly centrifuge the pGEM^®-T or pGEM^®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube.

2. 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.

3. 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

1. Prepare LB/ampicillin/IPTG/X-Gal plates (see Composition of Solutions).

2. 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) has been observed to increase transformation efficiency. Tubes from some manufacturers bind DNA, thereby decreasing the colony number, and should be avoided.

3. Place the high-efficiency JM109 Competent Cells in an ice bath until just thawed (5 minutes). Mix cells by gently flicking the tube.

4. 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.

5. 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.

6. Add 950µl of room temperature SOC medium to the ligation reaction transformations and 900µl to the tranformation control tube. Incubate for 1.5 hours at 37°C with shaking (~150rpm).

7. Plate 100µl of each transformation culture onto duplicate LB/ampicillin/IPTG/X-Gal plates. For the transformation control, a 1:10 dilution with SOC is recommended prior to plating.

8. 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 experiment 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

Screening of Transformants

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 β-galactosidase α peptide. Recombinant clones can usually be identified by color 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 that of colony PCR. It involves isolating the plasmid DNA from liquid cultures of individual E. coli colonies, performing the 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)

1. 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.

2. Boil for 10 minutes to break open the bacterial cell wall and release the DNA.

3. Centrifuge at 16,000 × g for 5 minutes to pellet the cell debris.

4. Use 5µl of the supernatant in a 50µl amplification reaction (see Table 13.2 for a sample reaction).

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.

5. 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.

¹Annealing temperature should be optimized for each primer set based on the primer melting temperature (T_m). 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.

6. Remove an aliquot of the completed PCR and analyze by agarose gel electrophoresis for the appropriate size product, which indicates the correct insert is present in the clone.

7. 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.

Subcloning

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, using the A overhangs to clone into another 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 Plasmid Subcloning and Ligation FAQ and the Subcloning Notebook.

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Cloning Protocol for the Flexi^® Vector Systems

PCR Primer Design, Amplification and Cleanup

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 fusion 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.

Restriction Digest of PCR Product and Acceptor Flexi^® Vector

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

1. 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.

2. Assemble the following reaction components to cut the PCR product with SgfI and PmeI.

3. Assemble the following reaction components to cut the acceptor Flexi^® Vector with SgfI and PmeI.

Note: Take care when pipetting solutions that contain glycerol, such as the Flexi^® Enzyme Blend, because small volumes are difficult to pipet accurately.

4. Incubate both reactions (Steps 2 and 3) at 37°C for 30 minutes.

5. 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.

6. Directly purify the digested PCR product using the Wizard^® SV Gel and PCR Cleanup System (Cat.# A9281).

Ligation of PCR Product and Acceptor Flexi^® Vector

1. Assemble the following reaction components:

2. 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.

Screening Clones with SgfI and PmeI

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)

• 10X Flexi^® Enzyme Blend (SgfI and PmeI; Cat.# R1851, R1852)
• Blue/Orange 6X Loading Dye (Cat.# G1881)
• plasmid isolated from colonies

1. Chill reaction components and reaction tubes or plates.

2. Prepare a master mix by combining the components listed below. Increase volumes proportionately depending on the number of reactions.

3. Add 15µl of master mix to 5µl (200–500ng) of plasmid DNA. Mix thoroughly by pipetting.

4. Incubate for 2 hours at 37°C.

5. Add 5µl of loading dye (Blue/Orange Loading Dye, 6X, Cat.# G1881). Incubate at 65°C for 10 minutes.

6. Load 20µl of the reaction onto a 1% agarose gel and separate fragments by electrophoresis. Visualize the fragments by ethidium bromide staining.

Transfer of Protein-Coding Region

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) or Single Step (KRX) Competent Cells (Cat.# L3001)]
• LB plates supplemented with the appropriate antibiotic at the appropriate concentration used (see Composition of Solutions and Figure 13.5)

1. 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 Protocols and Applications Guide chapter on DNA Purification]. Adjust the volume, so the final DNA concentration is 50–100ng/µl.

2. Assemble the following reaction components to cut the Flexi^® Vectors:

Note: Take care when pipetting solutions that contain glycerol, such as the Flexi^® Enzyme Blend, because small volumes are difficult to pipet accurately.

3. Incubate at 37°C for 15–30 minutes.

4. 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.

5. Assemble the following ligation reaction components:

6. Incubate at room temperature for 1 hour.

7. 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, as appropriate for the acceptor Flexi^® Vector. See
   Figure 13.5 for a list of antibiotic-resistance genes carried on the various vectors.

8. Screen at least 4 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 the insert can be cloned into other Flexi^® Vectors.

   Screen at least 8 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 because of 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 two culture plates for overnight growth in media: one with ampicillin and the other with kanamycin. Colonies containing the clone of interest will grow only in the antibiotic associated with the acceptor plasmid.