Thanks for the suggestions about cloning my cDNA in to expression vector. At the end with positive colonies, colony PCR worked quite well and I could easily discriminate false positive and positive colonies.
Now the problem. I used the mRNA coming from a donor to build up the insert to express the protein in a cell line. I would like to compare full lenght protein function against a shorter form of this protein.
I did few sequences but I need to do at least 7 sequences for each clone to cover the entire sequence of the insert. I noticed there are from 4 to 5 differences in nucleotide in around 2000 nucleotides. Since I used a nested PCR (with 20 cycles for the I round using external primers and 35 cycles using internal primers both with proof reading polymerase) to get the band of interest and probably there will be no clone 100% equal to my sequence of reference.....what should I do in order to select the clone I want to carry on with the analysis? I' m looking for clones that are without nucleotide differences, but I guess will be really hard to find one!!
Edited by micky74, 18 May 2009 - 05:01 AM.