|AccuPrime™ GC-Rich DNA Polymerase||100 μl
|5X AccuPrime™ GC-Rich Buffer A
|5X AccuPrime™ GC-Rich Buffer B
||1 ml||1 ml|
One unit of enzyme is the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 74°C.
Enzyme Storage Buffer
2 U/μl in 50-mM Tris-HCl (pH 8.0), 100-mM KCl, 1-mM Dithiothreitol (DTT), 0.1-mM EDTA, 50% Glycerol, and 0.1% Triton® X-100
5X AccuPrime™ GC-Rich Buffer
Buffer A and B differ in their concentration of MgSO4 and enhancers.
Key components are:
300-mM Tris-HCl (pH 9.2), MgSO4 at 10 mM (Buffer A) or 7.5 mM (Buffer B), 150-mM NaCl, 1-mM dGTP, 1-mM dATP, 1-mM dTTP, 1-mM dCTP, thermostable AccuPrime™ proteins, and enhancers
The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available here, and is searchable by product lot number, which is printed on each box.
Recommendations and Guidelines
Template: Use 5–100 ng genomic DNA or plasmid DNA, or 10–100 ng cDNA or bacteriphage lambda DNA
Primers: Use ≥50 ng each primer per 25-μl reaction. A Tm of 65–70°C is optimal for most applications. Primer design is one of the most important factors in successful PCR. We recommend using the OligoPerfect™ Designer, available at here.
Buffers: In general, we recommend using Buffer A for GC-rich genomic DNA targets and Buffer B for non-GC-rich genomic DNA, cDNA, and plasmids. Also use Buffer B if you find that Buffer A is inhibitory with your genomic targets.
Magnesium: MgSO4 is included in Buffer A at a final concentration of 2 mM and Buffer B at 1.5 mM. For some targets, more Mg2+ may be required; use the 50-mM MgSO4 provided in the kit to prepare a titration from 2 mM to 4 mM (final concentration) in 0.25-mM increments.
Reaction: Take appropriate precautions to avoid cross-contamination of DNA between reactions. Ideally, amplification reactions should be assembled in a DNA-free environment. Use of aerosol-resistant barrier tips is recommended.
Add components in the following order to each reaction vessel. Prepare a master mix for multiple reactions to enable accurate pipetting.
DNA template (above) x μl
Sense primer (10 μM) 0.5 μl
Anti-sense primer (10 μM) 0.5 μl
5X Buffer A or B 5 μl
AccuPrime™ GC-Rich DNA Polymerase (2 U/μl)* 0.5 μl
Sterile water to 25 μl
*Up to 2 U of enzyme (1 μl) may be added for difficult templates.
Cap/seal the reaction vessels and flick with your finger for several seconds to mix.
Program the thermal cycler as follows. Note that the annealing temperature will vary depending on the Tm of your primers. The optimal annealing temperature is typically 5°C below the Tm of the primers.
Step Temp (GC-rich
Denaturation 95°C 3 min
Denaturation 95°C 30 sec
55–65°C (5°C < Tm) 30 sec
25-30 Extension 72°C 1 min/kb
25-30 Final Extension
Maintain the reaction at 4°C after cycling. The samples can be stored at –20°C until use. Analyze 5–10 μl of sample by agarose gel electrophoresis.