I am trying to clone a chemically synthesised gene (1092 bp) into a expression vector called pET28a+ vector (5.3 kb). After PCR amplification of my gene of interest from the chemically synthesised plasmid I have double digested my gene with 2 different restriction enzymes: Nde I & Xho I. similarly I have digested my vector with the same enzymes and eluted both of them through gel extraction kit.
After this I did ligation in 1:1 insert:vector ratio and incubated overnight at 4 degrees.After tranformation in DH5-alpha comp cells, I got some colonies on LA plates but all of them were not able to grow in liquid culture. don't know the reason!
I also tried higher molar vector: insert ratios (like 1:3, 1:4 & 1:5) but still not able to get any colony.
can anyone plz help me out,
Hello
I will suggest you to check your ligation step. After ligation run the ligated product on agarose gel to confirm it.
Hi, I would pay attention to the digestion step. Even if you see your linearized plasmid on the gels maybe only one enzymes work well respect the other and you have an incomplete digestion (you perform a double digestion? How much plasmids you digest?).
As Mohammad suggested you ca use a positive control, it is strange that you did not have, at least, some false positive colonies (undigested pET28a+), it is very common. Check your DH5-alpha comp cells and you agar plates!
I am thankful to all of you for their respective answers.
Dear, Valeria I use approximately 1microgram of plasmid for double digestion. And I also checked for competent cells it is working fine. The colonies which I got are slow growing and some times I got colonies after transformation at room temperature and not at 37 degrees.
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