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Dear SEQanswer members
I've been using the SMARTseq2 protocol for some time now and have had some good sequencing results coming from it.
I have recently used the basis of the protocol to start amplifying whole genes of choice from the cDNA following the KAPA amplification steps and initial purification. After a gene specific round of amplification using a high fidelity polymerase, i send the products for standard sanger sequencing and it usually gives beautiful sequencing. However when cloning this PCR product i am finding that up 80% of my colonies have completely random SNPs all over the place.
These is no trend in these mutations, no common sites and usually not repeated. (To add i have used multiple polymerases for the second round amp and still the same effect.)
Has anyone experienced this before or know what might be going on?
Any ideas are most welcome. Thank you in advance.
Will
I've been using the SMARTseq2 protocol for some time now and have had some good sequencing results coming from it.
I have recently used the basis of the protocol to start amplifying whole genes of choice from the cDNA following the KAPA amplification steps and initial purification. After a gene specific round of amplification using a high fidelity polymerase, i send the products for standard sanger sequencing and it usually gives beautiful sequencing. However when cloning this PCR product i am finding that up 80% of my colonies have completely random SNPs all over the place.
These is no trend in these mutations, no common sites and usually not repeated. (To add i have used multiple polymerases for the second round amp and still the same effect.)
Has anyone experienced this before or know what might be going on?
Any ideas are most welcome. Thank you in advance.
Will