Screening for clones - (Jan/06/2012 )
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???
These empty colonies may be settalite colonies. Which selection antibiotic you are using ? May be u wait more than two day for appearence of colonies and antibiotic degraded.How mch plasmid DNA you are using for restriction digest. Increase the amount of DNA may be you get result.
This appears to be a self-ligation problem. During your ligation, you should have a self-ligation control (vector but no insert). Then do a transformation with this control the same as your experimental. If you are getting a plate full of colonies from the control, it will likely take a lot of screening to find a colony that has the insert. You could try SAP treatment of the vector to prevent self ligation. Also, try increasing the vector:insert ratio (1:5 even 1:10).