Transformation failure - (Oct/23/2008 )
Hello everyone,
I'm trying to clone a piece of DNA (200 bp), using a protocol that makes use of the A overhange generated during PCR amplification. (Qiagen PCR cloning kit)
Transformation is done as the following:
3 μl ligation-reaction mixture (Plasmid+ PCR product) is mixed with 30 μl competent cells, ice incubation for 5 minutes, heat shock at 42 C for 30 s (I've tried 1 min and 1.5 min as well), then ice incubation for 2-5 min.
The problem is that the competent cells seem to fail in taking up the plasmid (about 4 KB with the product). These E. coli cells are mutated for LacZ gene and are sensitive to Penicillin and Kanamycin.
The plasmid (obviously!) contains those.
What I get on the agar plates is the following:
X-gal: white colonies
Ampicillin: no colonies
Any suggestion for why the transformation fails?
Many thanks.
I'm trying to clone a piece of DNA (200 bp), using a protocol that makes use of the A overhange generated during PCR amplification. (Qiagen PCR cloning kit)
Transformation is done as the following:
3 μl ligation-reaction mixture (Plasmid+ PCR product) is mixed with 30 μl competent cells, ice incubation for 5 minutes, heat shock at 42 C for 30 s (I've tried 1 min and 1.5 min as well), then ice incubation for 2-5 min.
The problem is that the competent cells seem to fail in taking up the plasmid (about 4 KB with the product). These E. coli cells are mutated for LacZ gene and are sensitive to Penicillin and Kanamycin.
The plasmid (obviously!) contains those.
What I get on the agar plates is the following:
X-gal: white colonies
Ampicillin: no colonies
Any suggestion for why the transformation fails?
Many thanks.
Don't you also have some colonies which are pale blue ? If it's the case I would test them you never know. For failure of the cloning there's so much reason possible that it's hard to select one
How do you purify your PCR product after the PCR reaction ?
What is the ratio beetween plasmid and insert ?