Cloning in pCR2.1 plasmid - (Aug/09/2010 )

I have done a subcloning for the heavy chain of STRO1 antibody into TA plasmid .The sequencing has shown that the insert was not antibody but something else.I have used the right primers and I dont understand how is it possible that thez could have amplified something else.Is there anybody to who the same thing happened?.I have tought to blast the sequence to know what is in the vector but the plasmid has a larger number of bases and it will obviously interfere.Anyone has a suggestion ?

what kind of template did you use to amply the insert?

If you used genomic DNA, did you blast your primers against the genomic data base to see if the primers bind any other site aside from your gene of interest?

I used cDNA for the heavy chain and DNA for the light chain

Samira on Thu Aug 12 16:15:37 2010 said:

DNA, i am assuming it is genomic DNA?

DId you make the cDNA?

Did you do a blast search on the primers to make sure that there are no secondary binding sites?

What is the tm of your primers?

I dont know the tm for these primers.However, I am looking now to understand what the primers amplified and I did a blast serach but it is the plasmid sequence whihc comes as there are more than 1000 kb so how can I what the primers amplified and why did do that?please reply me as soon as you can.Thanks

Samira on Mon Aug 9 13:54:18 2010 said:

How exactly did you do this?

Samira on Mon Aug 9 13:54:18 2010 said:

I'm sure you used the right primers, however, your assumption that nothing else could have been amplified is likely incorrect.

Samira on Mon Aug 9 13:54:18 2010 said:

If you have a sequence that includes both vector and insert, you should be able to easily identify your insert sequence and isolate it for BLAST analysis. What is it you've cloned?

I have cloned the heavy chain of STRO1 antibody.How can I identify the sequence of the insert which is not the one that I was supposed to find?

Basically I amplified the cDNA with primers that my supervisor designed