lambda dna extraction

shantaram bharadwaj sb at biotech.ssf.ernet.in
Tue Apr 12 09:37:22 EST 1994


Subject:   Lambda DNA Extraction

I have been trying to prepare large amounts of lambda fix DNA.The lysate 
titre is very high.  After removal of cell debris, the phage is ppted with 
NaCl and PEG,  dissolved in TM buffer and PEG is removed by chloroform 
extraction.  I then purify phage using an ion exchange column.Phage yields 
are quite good. I have tried  1. Lysis of phage heads with phenol 2. Lysis 
with SDS and Proteinase K.The second method gives me better DNA yield than 
the first. The phage band looks intact and sharp on a gel, and there is no 
contamination withE.coli chromosomal DNA.  But it is resistant to restriction 
digestion.I have tried cutting with enzymes like Xho1,EcoR1, HindIII and 
BamH1 and there is no digestion.  There is no degradation since the phage 
band looksintact, even after long time incubation with the res.enzyme.  
There is no problem with the enzymes.  This is in spite of the follwing :
1 . I do extensive phenol:chloroform after lysis. 2.  I dialyse the DNA to 
remove SDS.  3. I put it through a column to remove phenol.
4.  Since ProteinaseK buffer has high amounts of EDTA, I use sod.acetate
    pH 7.0 for ethanol precipitation. (Ref.  Maniatis).
5.  I have used a mcr- E.coli host (SRB) to propogate phage. 
Currently,  our ultracentrifuge is out of shape, but earlier, a colleague   
of mine used the CsCl method to purify phage and SDS- Proteinase K method 
for DNA extraction. This DNA was digestible but there was problem in 
ligation.  Please send me sugesstions as to how I can overcome this problem.  
Has anybody else faced this problem?  If so, how did you overcome it?
Thanks.                                 Vasudha

-- 
Shantaram Bharadwaj
sb at biotech.ssf.ernet.in

-- 
Shantaram Bharadwaj
sb at biotech.ssf.ernet.in




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