Most of the human genes are GC rich, and its difficult sometimes to design primers in such regions. If any one has had the same problem and found a solution, please explain to me, how to sort it out.
Regards,
Dinesh
I amplify last week promoter with 99% GC (sliding window size = 42 bp) with KAPA HiFi Polymerase with the the GC Buffer. As the GC rich regions are hard to melt, you can increase the denaturating temperature to 98 °C, but note that not every polymerase can handle it. When designing primers, they should a have a high Tm (>65 °C). You can also design the primers in such a way that the Tm is the same like the elongation temperature (normally 72 °C), so you can do a "2-step" PCR. Beside KAPA HiFi, the Q5 Polymerase from NEB is also very good for GC rich templates. Both Polymerase are engineered (KAPA: by engineering the Pol itself, Q5 by fusing it to a DNA affinity domain Sso7d) that they have a very high processivity meaning that they have a high affinity for DNA and can amplify "faster" (1 kbp in 30 s).
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