Hi!
I hope you guys are doing well.
I am doing colony PCR for confirmation of positive transformation. After colony PCR I got the multiple bands on agarose gel. The bands that appear on the gel are neither equal to the size of the plasmid (5.76 kb) nor the size of the insert (2 kb).
I am unable to figure out whether the transformation was successful.
Please give your valuable suggestion.
Hello,
How did you select the positives (antibiotics or lacZ)?
The bands that appeared could also be due to parasitic amplification on the genome of the bacteria, so I advise you to carry out a PCR on the colony of your bacteria before transformation to see if there is still this amplification. Afterwards you also have to be sure that your selection method is good, perhaps you have selected false positives.
Hello Sofiane,
I used pQE trisystem vector that is Amp resistance. First I transformed undigested native plasmid to DH5-alfa comp cell and did the plasmid purification for restriction digestion with BglII and EcoRI from NEB in 3.1 buffer. Also digested the PCR product of target DNA and used for ligation with ligase from Takara.
ok so you selected according to the resistance or not of the transformed cells, did you obtain a lot of colonies? If this is the case, this potentially means that your antibiotic is not working and therefore even empty host cells are present.
Also, you do not have a way to phenotypically differentiate between cells which received the empty vector and those which received vector + DNA insert.
I got around 20-30 colonies after transformation of vector+insert.
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