I need to clone a full cDNA for protein expression (1782bp) and I have primers tagged with enzyme binding sites. I've tried to amplify the cDNA using different Tms and 5% DMSO in PureTaq Ready-to-Go PCR beads (GE) with no results. Now I'll try ramp PCR and, if necessary, SOE-PCR, but it's my first time. Any suggestions to help me?
http://link.springer.com/content/pdf/10.1007%2Fs10529-011-0656-y.pdf
Two enzymes that are very good at amplifying almost anything are:
-Phusion from Thermo Scientific with GC buffer (not the HF mentioned below):
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-hot-start-ii-high-fidelity-dna-polymerase/
-PrimeStar with GC from Takara (not the HF mentioned below):
http://www.clontech.com/takara/US/Products/PCR_Products/High_Fidelity_PCR/PrimeSTAR_HS_with_GC_Buffer
They are better than anything else I tried: Pfu (Strategene), Pfu Ultra (Stratagene), Herculase (Stratagene), HotStar HiFidelity (Qiagen), iProof (with HF or GC Buffer, BioRad), PrimeStar (with HF Buffer, not the GC mentioned above), Phusion (with HF Buffer, not the GC mentioned above), among others.
HotStarTaq DNA Polymerase (Qiagen) is a last resort since that one is potent, but not a proofreading enzyme.
Two enzymes that are very good at amplifying almost anything are:
-Phusion from Thermo Scientific with GC buffer (not the HF mentioned below):
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-hot-start-ii-high-fidelity-dna-polymerase/
-PrimeStar with GC from Takara (not the HF mentioned below):
http://www.clontech.com/takara/US/Products/PCR_Products/High_Fidelity_PCR/PrimeSTAR_HS_with_GC_Buffer
They are better than anything else I tried: Pfu (Strategene), Pfu Ultra (Stratagene), Herculase (Stratagene), HotStar HiFidelity (Qiagen), iProof (with HF or GC Buffer, BioRad), PrimeStar (with HF Buffer, not the GC mentioned above), Phusion (with HF Buffer, not the GC mentioned above), among others.
HotStarTaq DNA Polymerase (Qiagen) is a last resort since that one is potent, but not a proofreading enzyme.
How high is the GC content in your cDNA? We have had good results with Phusion as well (with the GC buffer), though our experience has mostly been with primers with high GC content (up to ~80%), not amplifying sequences with high GC content. A touchdown PCR program has also helped.
Hi Andrew! I have cDNA with 60 and 70% CG content, and the most part of CG is inside my primers, whose have very high melting temperatures. I'm trying DMSO and high Mg++ at this moment. What is the secret of Phusion? It cames with DMSO and has only 1.5mM MgCl2...
What is the Tm of your primers? (using the Tm calculator at http://www.thermoscientificbio.com/webtools/tmc/
It is OK for the primers to be shorter to keep the Tm reasonable. I have used primers with as little as 13 bp complementarity in order to keep the Tm low enough.
Also I assume that you have checked the primers for secondary structure and primer-dimer formation?
I have a big primer (27bp) because of the enzyme site at its 5'... Its Tm is 61,5C at complementary cDNA region and 85C at all. The amplicom has also a very high Tm (95C). My ramp PCR with 15% DMSO has "almost" a band. Maybe 20% DMSO could help... What did you think?
I would use Q5 DNA polymerase with annealing at 65°C in the first cycle and at 72°C in the rest. Denaturation at 98°C.
Alternatively, I would try 1, 1.5 and 2M betaine. Raising DMSO over 10% inhibits the reaction too much.
The problem can also be overcome by very long elongation times (10-15 min).
Good luck!
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