Help! Ligation of phophatased DNA...

[rcaspi at ucsd.edu]

Hello everybody

Are there any ligation wizards out there? If so, please help...
I am trying to ligate phophatased DNA to non-phosphatased DNA.
I have two different setups:
1. ligation of  phosphatased cut vector (pBlueScript) to non-phosphatased digested genomic DNA, in this case the ligation products are transformed to XL1 
Blue cells, plated on plates containing amp,Tc, and Xgal, and white colonies screened for a certain sequence by colony blots hybridization.
2. ligation of non-phosphatased digested cosmid (a modified pLAFR5) to partialy digested phosphatased genomic DNA. This ligation is to be packaged and 
transduced to prepare a library. Theoretically, only products of vector plus 30 kb fragments should be packaged.

Alas, whenever I try to ligate phosphatased DNA, it doesn't work. I tried both calf intestine alkaline phosphatase (CIAP) and shrimp alkaline phosphatase 
(SAP). I phenol-chloroform every reaction, chloroform it, precipitate and resuspend in clean distilled water before adding the ligase buffer and ligase, I keep 
the total concentration of DNA about 60 microgram per mililiter, I maintain a ratio of about 1.2 of vector:insert DNA, it never works...
I get good ligations if I don't use phosphatase, but the products are not what I need (self ligation of vector in the first case, concatamers of small DNA 
fragments in the later). 

Am I doing something wrong? Please answer directly to my email, if possible, as it is only too easy to miss a response in the news group.
Thanks,
-- 
Ron Caspi
Scripps Institution of Oceanography      Phone (619)534-0638
La Jolla  CA  92093-0202                 Fax   (619)534-7313