Screening for clonesNo insert relaese
Posted 05 January 2012 - 10:24 PM
I have been trying to clone a 375 bp insert into the PQE 30 vector Initially I was not getting any colonies on the LB with ampicillin plates (comp cell prepared by cacl2 method e. coli DH5) later I used comp cell prepared by friends by TSS method in that i got some colonies but when i screened them by RE digestion of plasmid isolated by alkaline lysis method with bamHi and PSTI I could not see any insert but there is only band of vector only
the primers which i have used are self designed and there is also problem with them that they amplify the gene and produce amplicon of the same size from simple E. coli DH5 cells too
so the question of colony PCR is ruled out for me can anyone suggest what i can do to confirm the insert or how to screen for positive clones
also suggest me why these clones with empty vectors are comming???
Posted 22 January 2012 - 12:11 PM
Posted 26 January 2012 - 11:30 AM