Standard sequencing primers - how to find which sequencing primers bind to my DNA? (Oct/06/2008 )
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.
Thanks in advance!
Correct me if I have misunderstood your situation
You have a gene encoding a protein which has been inserted into a plasmid. Now you would like to sequence the DNA outside this gene. You would like to sequence the plasmid vector itself, perhaps the promoter that is expressing your gene, or to ascertain the restriction sites that surround your gene.
Well, the simplest thing to do would be to buy/make/design primers that point outwards from your gene, towards the vector. These primers would easily sequence this unknown sequence. Most standard primers such as M13, T7 primers point inward towards the gene being expressed. They are designed to sequence the insert not the vector backbone. So I am uncertain how useful they would be.
Alternatively you can contact the people who build this plasmid and they should be able to provide you with the sequence map of the plasmid.
Thanks a lot!
You understood the situation correctly. Yesterday a friend of mine gave me similar advice and I think you guys are right and I have to make primers myself.
Anyway it seems to be an interesting general question to me if you can somehow find which standard primers (or primers that you already have) can bind to your DNA. I tried using pDRAW32 program that allows you to make a library of primers but I fell into troubles with creation of this library.
The most important thing is that I know what to do now.