Sequencing very short (60-80bp) amplicons ? - (Mar/26/2009 )
I have to know the sequence of a very short amplicon which are 60-80 bp in size.
As you know, capillary sequencers can only read regions after ~50 bp of the pimer binding site.
I found that pyrosequencing can read bases adjacent to the pimer binding sites, but our lab does not have any pyrosequencer
(We only have ABI sequencers).
And cloning does not seem to be easy for us.
Do you know any easy way to sequence such short amplicons?
I'll appreciate any comments.
If you are willing to look at the trace file and are good, you can see sequence from about 5 bp after the primer ends. So, you should be able to get sequence about 25 bp from the end of the product. By sequencing in both directions, you can get full sequence.
Alternatively, you can TA or Topo TA clone the fragment into a vector and use a primer to the vector for sequencing.
I suggest to review this page:
May be you can send your PCR product for pyrosequencing at UCLA
Another alternative is to add a tail to both primers
I agree with phage434 -- the way I'd approach this is to TA clone it, then sequence it from the vector.