Is it possible to get good PCR product if GC content of forward & reverse primer is 32% & 35% respectively. Tm of forward and reverse primer is 51.1 & 53.2o C respectively?
yes the likelihood of a good product depends on whether the primers can find other places to anneal which are not the target that you want. If similar sequences to your primers do not exist in your target genome then you will get a good product with almost any primers. So if your target is plasmid or cdna then you have a much better chance of amplifying a clean product than if the target genome is human dna for instance. It also depends on the GC content of the amplimer and its size.....a high gc template will not amplify well with low annealing temperature primers
Yes, it is always possible to get PCR product with low %GC of primers. If you are unable to amplify, then increase MgCl2 conc. & lower the temperature of amplification cycle to 68℃
I don't think so one criteria for choosing primers that GC content must be between 40-60% so I think must design primers again
There is no inherent reason they won't work with a low GC content. As Paul Rutland said, if your primers are specific to just your target area, then they will work.
Try them and see if you get the desired product.
I have the same question: My organism has a AT content of 80%. I am trying to verify a knockout strain. The primers worked well for the wild type strain and produced a 1kb product. It is expected for KO strain, they will produce a 2.5kb product. However, there is no band for KO when I run the PCR with both KO and WT. Should I lower the anealing temperature? I am doing 95 degree 60s, 95 30s, 45 20 s, 68 180s, 68 5mins, 30 cycle.
cant you design short amplimer primers just going into the normal or mutant sequence as shorter pcrs work better than long ones but also it is best to ask this as a separate question as it will get seen by more, and the right, people
Thank you Dr. Rutland. Yes I do have primers to test the hybridized part of the insertion. It will be more convincing if I have the comparison of the entire gene product between wild type and knockout. Yes it is a good idea to start a separate question.
Paul Rutland how do I deal with the primers having GC content 66-68%? Even if I used DMSO/glycerol what concentration and volume should be used?
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