Reagents & Solutions
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Characterization of TNT® T7 Quick for PCR DNA: Template
Purity
PCR Product Purification and In Vitro Translation
Aliquots (50µl) of each APC PCR were purified using the Wizard®
PCR Preps DNA Purification System* (Cat.# A7170, A2180) and
eluted in 50µl Nuclease-Free Water (Cat.# P1193). Assuming
96–99% yield (Technical
Bulletin #TB118), the PCR product concentration should be approximately equal before
and after purification. Aliquots of either unpurified or purified APC PCR
products (5µl) were then expressed in vitro using TNT® T7
Quick for PCR DNA*. The amount of radioactivity in each band was
quantitated by phosphorimaging. Figure
7 shows that, for the PCR products tested, purification is not necessary.
Ribo m7G Cap Analog
The inclusion or contamination of in vitro translation reactions with cap analog has
been theorized to inhibit translation by removing eIF-4 (eukaryotic initiation factor 4)
from the reaction. eIF-4 is necessary for
translation initiation as it recognizes and binds to the 5´ cap of mRNA and acts as a
binding site for other necessary initiation factors (6). The
T7 Luciferase Control DNA* (Cat.# L4821)
was linearized to mimic a linear PCR product. Linearized
T7 Luciferase Control DNA (500ng) was transcribed and translated in the TNT®
T7 Quick for PCR DNA system in the presence of 0, 8, 16, 80, 160, or 800µM Ribo m7G
Cap Analog (Cat.#
P1712). As shown in Figure 8, even 800µM CAP Analog did
not appear to inhibit expression of luciferase protein. It
is possible that higher levels of CAP Analog or lower amounts of template DNA will reduce
translation.
PCR Additives (Betaine, Glycerol, DMSO)
Many amplification targets require special PCR additives for
amplification success. These include reagents
such as betaine, glycerol and DMSO. To
investigate whether such PCR additives would inhibit expression using TNT®
T7 Quick for PCR DNA, linearized Luciferase T7 Control DNA was transcribed and translated
in the presence of increasing amounts of betaine, glycerol or DMSO. The results shown in Figure 9 demonstrate that significant
inhibition of luciferase protein expression is only seen at 2% DMSO, and some inhibition
is seen with 1.6% glycerol. Betaine did not effect
expression, even at a 100mM final concentration. Thus
the TNT® T7 Quick for PCR DNA system seems robust in terms of
carryover of PCR additives into the transcription/translation reaction.
(a)The method of in vitro expression cloning is covered
by U.S. Pat. No. 5,654,150 assigned to the President and Fellows of Harvard College.
(b)The PCR process is covered by patents issued and
applicable in certain countries. Promega does not encourage or support the unauthorized or
unlicensed use of the PCR process.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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Figure
7. Effect of PCR product purification on expression in TNT® T7 Quick for PCR DNA. |
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Figure
8. Effect of Ribo m7G Cap Analog on the expression of luciferase protein in TNT® T7 Quick
for PCR DNA. |
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Figure
9. Effect of various PCR additives on luciferase expression using TNT® T7 Quick for PCR
DNA. |
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