I am working on phylogeny of fungi. I amplify ITS region and get sequences. However the sequences are consistently bad nowadays. I didn't resolve this problem? I can not find the cause of the bad sequence.
My DNA was very clear and pure. Amplification of the region was successful and band profile was clear. Why the sequences are bad???
can you attach a raw sequencing .abi file please Aysenur.They have much more information than just the electropherogram
There may be quite a few reasons -
1. As Mr. Anjan told, DNA concentration decreases during cleaning up, so the elution process has to be performed with utmost care.
2. DNA quality and concentration both decreases if the time period between elution and sequencing is long enough...even a few days make difference. It is advised to directly go for sequencing following elution.
3. It is not clear whether the problem you are facing throughout or from a few days, there might be some issues with the sequencer itself.
thank you for the sequence Aysenur. Your loading and run conditions are good and the capillary acrylamide well in date.
The signal intensity of your sequencing starts at 1100 and drops quickly to 300-400 with a background of 8-11 so you should be able to read a single sequence well under these conditions but actually you only have 219 readable bases in the sequence. You can get this effect most often by having 2 or more sequences present and from the number of unreadable bases I think that either you have not got rid of all of your pcr primers so are reading the sequence from both ends or you have more than one sequence ( gel band) in your sequencing mixture. Was it a clean Product on the checker gel?.
The speed at which the signal intensity dropped may indicate some primer dimer present depleting the sequencing mix but I think that this is not the main problem.
Did you only sequence in one direction?. The homopolymer runs are quite clean which may indicate that your sequencing oligo is one base shrt as well as mainly full length. If you have only sequenced in one direction then try sequencing with the other primer, I am away for 9 days so will not be able to discuss this further. Good luck
Paul
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