Iunintentionally, I have used 1 micro gram , which is know to be too much, of my PET-M41.IgT plasmid to transform JM109 e.coli strain, and that eventually results in colonies on LB-kanamycin agar and those colonies were positive in colony PCR for my plasmid. While using a lower conc. Of my plasmid (23-38 ng of my plasmid resulted also in colonies on my LB-kanamycin agar but this time they were negative in colony PCR test? Is there any justification to this.
The plasmid which used in the first transformation ( high conc.) was obtained from a colleague who designed it, while in the 2nd time (low conc) it was the plasmid that I have amplified from the original stock in JM 109 e.coli strain ( positive for colony PCR).
Hi, have you check your Plasmid, which you have amplfified with a resctriction analysis, so that you know that the plasmid is still fine? We got always better results with "low concentration" of the plasmids when we did a transformation. Problems have occur when the antibiotic concentration was not fine, or our plasmid conctruct was not okay anymore.
My colony PCR was never affect by the plasmid concentration which I use for transformation.
As far as I know, colony PCR is very sensitive even with nano grams of DNA present. Maybe you can set up a control plate, streaked with non-transformed bacterial cells. If there are too many colonies in this negative control plate, then the problem is with the LB/Kan plate. If control plate has far less colonies, or no colony at all, pick more colonies to do PCR.
Yes Urda the plasmid is fine, as 1ul of the plasmid i a PCR reaction showed +ve for my insert.
Yes Yating,
Non-transformed cells on lb+kanamycin showed no colonies at all.
Hi, Ali
This is a good point, and yes I have purified the plasmid from the positive colonies and tested the conc. with nano drop and a further PCR analysis showed that the palsmid is fine and has my insert.
As I have said before, I got this pasmid from a colleague, I don't know about the purity of the (plasmid+ insert) in the tube in comparsion to the -ve plasmid, could it be that the bacteria that grow on the kanamyicn supplied agar did engulph the plasmid without insert (-ve one)?
Thank you veryy much dear Ali, I really appreciate your contribution and will consider both of your tips :) :).
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