I am trying to clone a 1.2kb gene in an integration vector (5.1kb) which has two antibiotic resistance sites (Ampicilin and Chloramphenicol) and an integration site with the promoter and terminator region of a house keeping gene and an Nde1 locus in between the promoter and terminator region where the desired gene can be inserted and expressed constitutively (when integrated into gram positive bacterium). The vector can replicate in E.coli.
I have added Nde site at both sides of my gene (by PCR); digested the (purified and concentrated) PCR fragment by Nde1, gel purified the fragment and checked again for the correct size.I have also digested the vector with the same enzyme (Nde1), purified (using Qiagen kit), treated the eluate with alkaline phosphatase, gel purified the product and ligated the insert with the vector using T4 DNA ligase (Roche, quick ligation buffer) at RT for 20 mins. I checked my ligation reaction (by PCR, using vecor specific forward and insert specific reverse primer). Then I transformed the ligated product (both chemically and electroporatically) using Top10 and DH5 cells and plated them on AMP+Chloramphenicol plates. Next day, I got positive colonies on all of the plates (with chemically/electroporatically transformed samples). I screened 40 colonies (10 from each plate) and failed to get any positive colonies by PCR screening. However, when I reduced the annealing temp of the PCR reaction (from 53 degree to 44 degree) and ran the PCR product(with vector specific and insert specific primers) in gel, most of the colonies looked positive with the correct target size along with some nonspecific bands. However, I failed to digest the apparently positive samples with any of the vector specific restriction enzymes. I also tried to sequence the plasmids (using vector specific primer) without any success. I have repeated the ligation and transformation parts several times. The result was the same. Colonies on double antibiotic selection plates, but no result with restriction digestion/sequencing. Please help me with any kind of suggestion.
Thanks a lot in advance.
0
No replies to this topic
