I have a Crispr/Cas9 edited bulk population and I can briefly check the indel ratios in the bulk population using Sanger sequencing. After cloning single cells, I wanted to check the indel types in different clones. The normal PCR product size is 531 bp but in some clones, the reading stops at around 300 bp (right after the cutting site). I can still have the indel information from those readings but I wonder why the reaction stops. Is somehow the polymerase slipping? I checked the creation of potential repetitive sequences after the editing in each clone, but it does not seem like the case.
Thank you,
Can you attach one of the short sequence .abi sequence files and ideally a normal sequence .abi file please?
Maybe it belongs to template concentration or contamination in colonies. Also, you can use the other primer from the opposite side of your target fragment
As Paul Rutland suggested it would be helpful to see the files to better interpret what is happening. But if the sequence reads stop right at the place where you expect the indels it might be that you still have mixed populations and the reads stop because the sequence diverges after that point. Looking carefully at the files will tell you whether there is no sequence information or it is just garbled due to mixed bases at each position.
Paul Rutland and Michael J. Benedik
You are right, it would be easier with the files. Please see the files attached. As you see, non-edited cells give good reading but in some edited clones (not all though), right after the cutting site, we start seeing double-peaks till the end.
It looks to me that you have mixed clones in both 3-58 and 7-58 which are identical for the first 300 nts and then they diverge. I would guess each has two sequences. You might actually be able to figure out the sequences of both DNA present in each DNA prep if you know the underlying sequence and do some detective work since there are only 2 bases read out at each position. Alternately you could retransform that DNA prep and pick a few independent transformants and resequence them.
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