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Focus: in vitro transcription/translation using PCR templates
Compatibility of GoTaq™ DNA Polymerase-Amplified PCR Products with TnT®
T7 Quick for PCR DNA
This
article describes use of PCR products generated with GoTaq™ DNA Polymerase
for in vitro transcription/translation
experiments using the TnT®
T7 Quick for PCR DNA system (Cat.#
L5540). DNA fragments generated using GoTaq™ DNA Polymerase
performed well with this in vitro expression system. |
By Natalie Betz, Ph.D.
Promega Corporation
Introduction
GoTaq™ DNA Polymerase(a) is a novel DNA polymerase formulation
containing native Taq DNA polymerase in a proprietary formulation based
on PCR Master Mix (Cat.#
M7502). GoTaq™ DNA Polymerase is supplied with 5X Green
GoTaq™ Reaction Buffer and 5X Colorless GoTaq™ Reaction Buffer. The Green
Reaction Buffer contains two dyes (a blue dye and a yellow dye) that separate
during electrophoresis to monitor migration progress. The buffer also contains a
compound that increases the density of the sample, so it will sink into the well
of the agarose gel, allowing reactions to be directly loaded onto an agarose gel
without the need for loading dye (1).
The purpose of this experiment was to verify that PCR products generated
using GoTaq™ DNA Polymerase can be used as template DNA in an
in vitro expression system (TnT®
T7 Quick for PCR DNA; Cat.#
L5540). The APC Segment 3 PCR product was
chosen as the target DNA. The product of the APC segment 3 gene is more
difficult to express compared to firefly luciferase and so may provide a
more sensitive measure of inhibitory substances that may carry over from PCR.
Mutations in the APC gene have been linked to familial adenomatous polyposis.
Disease-associated mutations result in a truncated protein product and tend to
be clustered in a small region, designated the mutation cluster region (MCR);
(2).
Methods and Results
Procedures were performed according to the methods
provided in the GoTaq™ DNA
Polymerase Product Information Sheet and the TnT®
T7 Quick for PCR DNA Technical Manual (#TM235)
(3,4).
The APC segment 3 PCR product was generated from wildtype Human Genomic DNA (Cat.#
G3041) in three separate PCR amplifications using either Tfl DNA Polymerase
(positive control), GoTaq™ DNA Polymerase in Green Reaction Buffer, or GoTaq™
DNA Polymerase in Colorless Reaction Buffer. The composition of the
amplification reactions is given in Table 1.
Table 1. Assembly of APC
Segment 3 PCR Amplifications. |
Reaction
Component |
Tfl
DNA Polymerase |
GoTaq™
Green
|
GoTaq™
Colorless |
Enzyme |
1µl |
0.25µl
|
0.25µl
|
Buffer |
5µl
5X AMV/Tfl Buffer |
5µl 5X
Green GoTaq™ Reaction Buffer1
|
5µl 5X Colorless GoTaq™ Reaction
Buffer1
|
dNTP Mix |
1µl |
1µl
|
1µl
|
25mM MgSO4 |
2µl
|
0µl
|
0µl
|
Primer Pair2
(50pmol each/µl) |
1µl |
1µl |
1µl |
| Human Genomic DNA
(270ng) |
1µl |
1µl |
1µl |
Nuclease-
Free Water |
39µl |
41.75µl |
41.75µl |
|
1Both Green and Colorless GoTaq™ Reaction Buffers
contain MgCl2 at a concentration
of 7.5mM, giving a final concentration of 1.5mM in the final reaction.
2Forward Primer: 5´-GGATCCTAATACGACTCACTATAGGAAC
AGACCACCATGCTTAAATATTCAGATGAGCAGTTGAA-3´
Reverse Primer: 5´-GAGCCTCATCTGTACTTCTGC-3´
|
The cycling conditions were as follows: 1 cycle of 94°C for 3 minutes;
35 cycles of 30 seconds at 94°C, 1 minute at 60°C, and 2 minutes at 72°C; 1
cycle of 7 minutes at 68°C; soak at 4°C. An aliquot of each reaction was
analyzed on an agarose gel. All amplification reactions produced the expected 2kb APC Segment 3 PCR product (data not shown). PCR products were
quantitated by PICOGREEN® analysis following the manufacturer's
directions (Molecular Probes). The PCR products ranged in concentration from ~3–16ng/μl.
Duplicate (50μl) in vitro protein expression reactions were assembled using
the TnT® T7 Quick for PCR DNA
system and
18ng of APC Segment 3 PCR
product. Following incubation at room
temperature for 90 minutes, each set of duplicate in vitro transcription/translation
reactions was pooled and an aliquot (3μl)
analyzed on a 4–12% NuPAGE® Bis-Tris gel. The gels were fixed, dried,
exposed to a phosphorimaging cassette overnight, and the amount of APC
Segment 3 protein produced was quantitated using the Molecular Dynamics STORM®
analysis system.
The results are shown in Figure 1. All of the APC Segment 3 PCR
products translated well, producing the ~80kDa APC segment 3 protein regardless of the enzyme or buffer used for PCR
(data not shown). Thus,
PCR product generated with GoTaq™ DNA Polymerase and either the Green or
Colorless GoTaq™ Reaction Buffer are compatible with in vitro translation
using the TnT® T7 Quick for PCR DNA
system.
References
- Glebs, A., Stencel, E. and Knoche, K. (2003) Introducing GoTaq™ DNA
Polymerase: Improved amplification with a choice of buffers. Promega Notes 83,
21–4.
- Kinzler, K.W. et al. (1991) Identification of FAP locus
genes from chromosome 5q21. Science, 253, 661–5.
- GoTaq™ DNA Polymerase Product Information Sheet, Promega Corporation.
- TnT®
T7 Quick for PCR DNA Technical Manual
#TM235, Promega Corporation.
(a)Certain applications of this product are covered by patents issued and applicable in certain countries.
Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a patent
license depending upon the particular application and country in which the product is used.
Products may be covered by pending or issued patents or may have certain
limitations on use. Please visit
our patent and trademark web page for more information.
GoTaq is a trademark of Promega Corporation. TnT
is a trademark of Promega Corporation and is registered with the U.S. Patent
and Trademark Office. NuPAGE is a registered trademark of Novel Experimental
Technology. Picogreen is a registered trademark of Molecular Probes, Inc. STORM
is a registered trademark of Amersham BioSciences, Ltd.
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Figure 1. In vitro
expression of APC segment 3 PCR products generated using Tfl or
GoTaq™ DNA Polymerase. |
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