Cloning in pCR2.1 plasmid
Posted 09 August 2010 - 05:54 AM
Posted 10 August 2010 - 05:10 PM
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?
Posted 12 August 2010 - 08:15 AM
Posted 14 August 2010 - 07:05 PM
I used cDNA for the heavy chain and DNA for the light chain
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?
Posted 15 August 2010 - 03:41 PM
Posted 15 August 2010 - 04:08 PM
I have done a subcloning for the heavy chain of STRO1 antibody into TA plasmid.
How exactly did you do this?
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.
I'm sure you used the right primers, however, your assumption that nothing else could have been amplified is likely incorrect.
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.
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?
Posted 16 August 2010 - 01:02 PM
Posted 16 August 2010 - 01:08 PM