Question about genomic library construction
bss194 at thunder
Thu Feb 27 06:22:31 EST 1997
Alexander N. Kukushkin (kan at mmcd.cyt.ras.spb.ru) wrote:
: > Hello everybody, I have some trouble in my project and ask for
: > your suggestion. I have constructed a genomic library for half year,
: > however, I still cannot clone any fraction of insert into the vector with
: > high efficiency. Here is the method I used: the vector pUC19 digested by
: > EcoRI and dephosphorylated by CIP (one hour), the insert was obtained by
: > EcoRI digestion of the genomic DNA and the digested DNA was separated by
: > eight fraction (the size from 0.5kb to 6.0kb). The vector and the insert
: > was ligated by T4 ligase at R/T for 2 to 4 hours. After transformation, I
: > can find normally ten to twenty colonies from the plate and after
: > analysis, only one recombinant can be found from 16 colonies. The
: > ligation efficency is very low, What should I do?
: > Wilson 8~O
: CIP may be serious reason for the low ligation efficiency.
I have to agree with Alex that this is one of the most likely problems. I
am replying because I've done two very similar clonings in the last
month, so it's very fresh in my mind:
First- If you have to use CIP try to use a cloned, bacterially expressed
one as they seem to cause less damage to the DNA ends (from
personal experience). Limit the incubation time to 30 min, rather
Second- Inactivate the CIP *really* well. I use a 30 min incubation at
55C in 0.1% SDS, 100ug/ml Proteinase K followed by phenol
extraction, then chloroform, then ethanol precipitate. Or you
could use shrimp AP which you can kill by just heating.
Third- Do controls such as- re-ligate and transform some non-CIPped pUC
to check your ligation efficiency, transform some uncut pUC to
check your competent cells, maybe transform some re-ligated
CIPped pUC to check your background level of uncut/undephos'd
Agarose contamination can upset T4 ligase, so you may want to check your
gel extraction method, but I would start with the CIP.
Keith James Ph.D. - k.james at bangor.ac.uk PGP 2.6.2i Key ID 469A9FA1
Biodegradation Group *Encrypt and Survive*
School of Biological Sciences Nightmare: Quake me up now!
University of Wales, Bangor, UK
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