how to get DNA sequences starting from the primer - (Apr/22/2007 )
Hi I am currently doing some sequencing, and I can only obtain a good reading at around 20-30bp away from my primers, so I was woundering how I can modify my protocol to obtain sequences starting from my primers.
Any suggestions will be great and thanks in advance!!
Short answer: you can't. Slightly longer answer: you can sometimes figure out what is happening by carefully looking at the trace file and manually calling bases in that region. You can help a little by taking care in the sequencing reaction cleanup to keep the short fragments. The real answer is to sequence in the other direction with a new primer, which you can derive from the sequence you already have.
When sequencing manually, we would label the primers rather than incorporating. Doing this would give all products lengths equal signal, and we could read the sequence from the first base beyond the primer.
If your DNA is incorporated in a plasmid, use the universal sequencing primers that should be in the plasmid sequence either side of the MCS.
beckman-coulter (or, at least, one of their sales reps) recommends the use of qiagen spin columns to clean up the reaction. this can allow you to read from ~5 bases from the primer.