DNA sequencing primers

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alex.bondar3
alex.bondar3's picture
DNA sequencing primers

Hello!

I have a basic question. I have a construct encoding a protein. I know DNA sequence of the protein but I don't know the sequence of the construct outside the protein encoding part. So I need to perform DNA sequencing outside the known DNA. The question is: How can I find if any standard DNA sequencing primers (like T3, T7 etc.) can bind the part of DNA sequence that I know? I would prefer to use standard primers instead of making them myself if I have a choice. Manual alignment of primers to DNA doesn't work because a have a list of app.
300 primers.

Any suggestions?

Cheers,
Alex

ryan_m
ryan_m's picture
I would not suggest you try

I would not suggest you try to find a primer that is already known that has some similarity to your insert. This is not likely to work (and it is more likely that you will later find out the primer has a perfect recognition site in the vector itself). If you know what the vector is, you should be able to find the sequence from the supplier of that vector. If not, you should be able to design a pair of primers that anneal to opposite strands within the insert (but are oriented to point away from one-another). This would allow you to produce an amplicon containing the whole vector. You could then use the same primers as sequencing primers and sequence either end of the cloning site of the vector (hopefully then you could recognize the vector and look up the remaining sequence).

alex.bondar3
alex.bondar3's picture
Thanks a lot!

Thanks a lot!

I will try to sequence the whole vector.

Cheers,
Alex