I did sequencing for my positive PCR product. The expected band size is 274bp.. but when i did sequencing to this PCR product (using only forward primer), the nucleotide sequence length was only 153bp although it is really my target (i did BLAST to this sequencing result)..
If it was 153bp length, then why was the band around 250bp size? What is the explanation for this?
Thank you..
Dear Lidya, most probably your sequence was not read completely, if you carry out sequencing by reverse primer also you can get full sequence of amplicon. Usually we face with this situation with long fragments. Your sequence is short, it must be read with only forward primer.
Another reason maybe related to your cycle sequencing reaction and labelling nucleotides.
Best regards.
I think Mert is correct. You cannot read the sequence under the primer and the sequence for the next 30-50 bases from the 3' end of the sequencing primer is poor and may not be base called so you will have to sequence in both direstions to get both ends of the sequence properly
Yes, the problem was during the sequencing, did you sequence two reactions, one with each primer and after assembled both reads? That can help.
Paul is correct, you need to sequence your PCR product from both directions and create a consensus sequence to get the entire length.
a further thought Lidya,
usually you cannot sequence under the primer or the next 30 bases but in your experiment you are not sequencing about 100 bases so the good quality sequencing is starting rather late. Sequencing kits are designed to sequence up to 1000 bases but you are only sequencing 250 bases so to get a better sequence starting closer to the primer I would use more template in the sequencing reaction and also more sequencing primer...say 5pmol and increase your template to 20ng
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