My possitive control for transformation with original plasmid worked well and obtained colonies.
Hello,
In this case, potentially, your recombinant DNA (Plasmid + insert) remained open, that is to say that your DNA ligase, did not make a connection between these 2 DNAs. An open plasmid cannot be transferred to the bacterium.
Also, are you sure that during the opening of your plasmid with the restriction enzymes, you didn't break your plasmid?
Sofiane Benyamina Thanx for your answer.
I am pretty sure that my plasmid not gets broken while opening with restriction enzymes.
In this case, there remains the possibility that the ligation did not work well and the plasmid remained open.
possibility 1: something went wrong with cloning: For checking this you can include all possible controls (a: was plasmid well cut? can you see definitely the all sites you wanted to cut are cut? same for fragment b) did purification of fragment/plasmid work? check on gel, not just measure concentration c) does ligation work? Have you recently performed successful ligation? Ligase and ligase buffer can deteriorate. You can cut and religate vector as a control or ligate with a smaller fragment or something u know works) If you have a lot of material u can run the ligation on gel also.
possibility 2 is that cloning went fine, but plasmid is either very large (this can give problems with not super competent cells) or your gene is somehow toxic. If you think your insert is very big and this could be an issue, you can try using competent cells with higher efficiency or e.g. electrocompetent cells or more DNA in general. Sometimes extending the recovery time after heat shock or electroshock a bit for large plasmids can help (i think). For toxicity, sometimes different strains could help or lower temperature for incubations.
- Badges
- Science topic