deletion during cloning - (Nov/13/2005 )

Hi all,

I got a deletion during my cloning.

FYi, i sequential digest my 12kb plasmid (KpnI & NotI) and ligate it with 30bp linker. I screened my colony by cutting it using RE that will ONLY recognise my linker. I got it!! But afterward i found out that my inserted plasmid have deletion in somewhere else by looking at the gel (approx. 1kb deleted).

Anybody encounter the deletion before yet with insert successfully go in? wat's the problem???


confused ,
cyk

I have seen this before but infortunataly cannot tell you why it happens. I have cloned fragments into pBluescript and gotten positive colonies, but when I have checked the size of the plasmid by gel, its smaller than the plasmid containing no insert!! I think it may be the clean up of the DNA. Did you gel purify the fragments and if so, did you run the gel with ethidium bromide and/or expose the DNA to UV? If the DNA is damaged, it may clone but then the damaged DNA may be cut out by the cells repair machinery. Don't know if this is the case, but it may be a possibility.

I'm having a similar problem cloning. Currently I'm trying to subclone mus cytokine DNA into pGEX vectors but with no luck. Everything looks fine up until screening inserts. Everything looks strange after screening colonies and RE rxn's. Even the plasmid DNA is either too small or too large. I'm guessing and hoping it's because I gel purified on a 312nm UV trans. But I've seen E. coli do odd things before with DNA. Has anyone else had problems with E. coli cutting up certain DNA sequences?

Regards

thanks for the reply.

i screened other colonies on the same plate and found that some of them are actually fine. I think i may have overdigested it and caused some of the plamid DNA started to nick at other part, hence causing the ECOLI to "repair" it. Do u think it is a valid reason?

cyk