Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Cloning done....sequence of the insert for the expression vector - (May/18/2009 )

Hi every one

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!!
Any suggestion?



I agree with you, it is sometimes impossible to find a perfect match.
For protein expression you can select for clones that show only silent mutations. If you find that all of your clones show the same amino acid change, consider that your reference sequence might be wrong... Those sequences are far from perfect, depending very much on individuals or mouse strains. From my experience, cloning by PCR gives you 1nt mismatch per 1kb. If you have more, it is more likely that your source sequence was already wrong.
If you desperately want to have the published sequence, you might need to do some site-directed mutagenesis.

Good luck!