I have a gene with GC content 83%. The Forward primer and Reverse primer GC contents are 73% and 63% respectively. I have tried DMSO concentration from 1-10% for optimization and found only faint amplification.
What is the size of the cDNA? For difficult to amplify genes, gene assembly using oligos or commercial gene synthesis are often good alternatives to PCR. If you are not modifying the sequence and it is already cloned into a plasmid, perhaps you can subclone it without PCR. Some more information in your post about what plasmids or DNA templates you are using and whether you are changing the cDNA sequence (for example, add a tag) would be useful to share.
If you want to spend the effort troubleshooting the PCR strategy, try a gradient of annealing temperatures in addition to the DMSO titration.
I made positive experience with betaine (1 M final conentration). Qiagen sells this stuff as Q-Solution with their PCR kits (and you can't get it solo) but Sigma offers a 5 M stock solution.
"Frackman, S.; Kobs, G.; Simpson, D. & Storts, D. Betaine and DMSO: Enhancing Agents for PCR Promega Notes, Promega Corporation, 1998, 65, 27"
Alternatively, you could use the Phusion Taq polymerase with the GC buffer.
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I agree with Philipp. You can use betaine even to 2-2.5 M. Normally, I use to buy it solid and then make my own solution stock…It is 1000x or more cheaper….Moreover, using a polymerase with strand displacement capacity such as Platinum Pfx improves further your PCR…
I have found that Accu pime GC rich Taq polymerase (from Invitrogen) works really well for GC rich amplication.
If the various specialized thermophilic polymerases don't work, you could easily assemble a 700bp cDNA from oligos or consider the gene synthesis route. While gene synthesis is a bit more costly, investing in purchasing various polymerase and the time of optimization may cost more in the long run.
Had the same problem and same experience like others here, cloning a gene with high GC content and making fusion constructs with the cloned gene. PCR failed under standard conditions (both with Phusion and Kapa Hifi), DMSO didn't improve the conditions, used the betaine solution from Sigma for cloning, worked beautifully, and also worked for all further PCRs with the same gene.
Phusion II (Hot Start with GC buffer) from Thermo Scientific or PrimeStar (Hot Start with GC Buffer) from Takara/Clontech are great working enzymes. Gene synthesis at less than 0.3 dollar/nucleotide is almost unbeatable, plus you can have the GC content and codon usage changed for optimal expression in your organism(s) of choice.
We use Q-solution in our PCR reaction, it's included with the Taq from Qiagen, and it works very well.
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