After I've done with cloning, there's positive growth in antibiotic plate but not in negative control.
Few potential colonies were picked and incubated in broth containing selective antibiotic and grow overnight (20 hours).
The plasmid extracted was quite low, about 20ng/ul.
I then use 5ul for pcr amplification and 10ul for RE digestion to confirm the presence of dna insert in the plasmid
PCR shows no sign of dna insert being amplified. While no any single band was found in gel after RE digestion.
I wonder what could potentailly went wrong.
1. Could it be the amount of transformed plasmid was too low for the confirmation tests above?
2. Could it be the the false positive growth in antibiotic selective plate?
3. Could it be the insert was unstable in the vector? But even if it's unstable, I should still see the band of vector after RE isn't it?
Please advise. Thanks.