Dear all,
I am planning to sequence (sanger sequencing) pcr products (around 300 bp) for SNPs detection. I was wondering which polymerase has the best effective/cost ratio for this purpose???
Thank you for your time
All taq polymerases are fine for producing a pcr product for sanger sequencing. All enzymes will make some incorporation errors but with Sanger sequencing the sequence at each base is the contribution from all the bases present so unless a change happened in the first few cycles of pcr the changed bases will be swamped by the signal from the correct bases at all positions if you sequence directly the pcr product. This does not apply if you are cloning first and then sequencing when only one sequence will be present.. It is more important to keep the annealing temperature of the pcr primers high as this will help any polymerase to be at iys most accurate. I would sequence with ABI kits diluted more than the instructions say as your amplimer is only 300 bases then once you have identified your SNPs if you have a lot of samples then a method like DCAPS or ARMS might be a cheaper option for identifying SNP bases
Any Taq polymerase. In PCR followed by Sanger sequencing (of rather small products) crucial is designing really good primers (without SNPs, specific, with an appropriate melting temperature).
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