Hello,
Last week I asked my PCR results and I send Sanger seq. Well, there is one colony including mutation, I guess. How to cleaning my sample for Sanger? The Sanger result belongs to the second sample in the PCR result. The base stated by S is the base I'm trying to create the mutation. G>C mutation.
can you attach an original .abi sequence file for this sequence and ideally a similar file for the normal sequence please.It is not clear if you have a high background or if the overlying sequence is caused by in insertion/deletion. Cloning the pcr sequence and resequencing may clarify the sequence. how did you clean the sequenced pcr band before sequencing it
Hi Merve,
what Paul said is highly possible, an insertion/deletion in your sample. to be fine with that, you could run the sanger sequencing from both ends (in reverse and forward) and see where the variant is, if it is...
all the best
fred
You have multiple bands on your gel, including the second PCR sample (from either the left or the right). I assume that means you did a gel excision and clean up before sequencing? Otherwise you will have overlapping sequence from two different PCR products.
I would perform a blastn search to check what the majority of sequences have in that region and whether your chromatogram matches. It looks like if you have a peak of C before GGG.. .
You can use forward and reverse primers to sequence from both ends. However, it's very clear that you need cleaner DNA samples as indicated by Katie. You can think of changing PCR parameters to get rid of unwanted bands.
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