Dear Colleagues and Mentors,
Hope you all are doing fine.
I'm trying to sequence a gene which has partially sequenced before. Now, our plan is extend the sequence or if possible get the whole sequence. I have used the partial sequence to design my primer. After running the common PCR and Gel electrophoresis, I have got band which was sent to sequence, but the sequence I received is not the one I expected. This is something weird for me.
Now, I'm asking help from you to solve the problem.
Have fun and cheers!!!!!!!
when you blast the sequence that you have produced is it anything to do with your gene or another sequence completely. If it is part of the unsequenced part of your gene how do you know that it is not your gene? Are there similar genes from different species that might help you design primers and do you know why the gene is only partially sequenced previously?
Dear Sarker,
as Rutland said, blast your sequence and decide whether it is your sequence or not. you can also, know from contiging the sequence with your reference sequence. Regarding the partial sequence, you may find the full length of the gene in ENSEMBLE or NCBI if not found, try to use its ortholog from the another species.
Something is missing here ? How to get the whole gene from a partail sequence unless you are doing a primer walk or using degenerative primers based on orthologs genes of closely related organisms?
However, try to clone the purified PCR product of your gene then search for your forward and reverse primers (if everything works fine, you will find your gene between the two primers)
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