Hi everyone,
I am doing a conventional cloning ligation procedure using double digestion (BamHI and EcoRI) using pGEX-6P-1 vector with my purified PCR product of 393bp. After ligation in 6:1 ratio, I cracked the colonies to find few band shift from empty vector where I further subject their purified plasmids/colonies to PCR transformation analysis with pGEX sequencing primers flanking both BamHI and EcoRI respectively. Firstly, why do I have so different band sizes from lane 3, 9, 13 and 23? Is the primer not specific or the vector has taken different bacterial genes? Secondly, lane A is my PCR product for size comparison while lane 23 is closest to my band size of interest. After sequencing PCr product from lane 23, the result was an E. coli YJ4 DNA sequences, how is this possible?
Thank you
Note: I used DH5 alpha for the transformation with Carbenicillin as selective marker.
It looks like something went wrong during your PCR as you could see some smears above and below your band of interest. What was the template you use for amplifying your PCR product ? Was it genomic DNA ?
As for testing your colonies try to use classical primers like T7 or T3 or if they are not present in the MCS use M13 reverse and forward primers
Well I check your plasmid map and it has only the PGEX sequencing primers so no way to test differently. As for the PCR on cDNA it's really weird that you get a E.coli contamination. You could send your PCR product for sequencing using the same primer you use for amplification and make sure that the PCR is really specific. Looking at your gel you must have more than enough to get a good sequence out of it
Dear Isiaka Muhammad and all others.
Let me ask you a few things about your project.
1 - Do you have background on cloning or just started to work with it? Just to know your background ...
2 - Have you checked if BamHI and EcoRI cut your product or plasmid in other places than the ones for cloning?
3 - Why did you choose to perform a ratio of 1:6 instead of 1:3, for example?
I will wait your answers in order to try to help you.
All the best.
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