Related Product Information
|Component||200 Rxns||1000 Rxns|
|AccuPrime™ GC-Rich DNA Polymerase||100 μl||500 μl|
|5X AccuPrime™ GC-Rich Buffer A||1 ml||5 ml|
|5X AccuPrime™ GC-Rich Buffer B||1 ml||5 ml|
|50-mM MgSO4||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 on our website at www.invitrogen.com/cofa, 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 www.invitrogen.com/oligos.
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
Time Cycle Denaturation 95°C 3 min 1 Denaturation 95°C 30 sec 25-30 Annealing
55–65°C (5°C < Tm) 30 sec 25-30 Extension 72°C 1 min/kb 25-30 Final Extension 72°C 10 min 1
- 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.