Question about genomic library construction

K.James 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.
: Alex

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 
	than 60.

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 
	pUC. 

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                                     
-------http://oracle.bangor.ac.uk/sbs/research/biodegradation/-------



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