PREPS:lambda DASH II/lambdasorb
hmmoss at MAIL.MED.CORNELL.EDU
Fri Jan 20 18:54:45 EST 1995
I was searching through the December "methods and reagents" section and
discovered your questions regarding lambda DASH II and LambdaSorb (Promega).
Although you might have already resoved your dilemmas, I thought it would
not hurt to write anyway.
I am working on clones isolated from a human genomic library propagated
in Lambda Dash II. I was interested in mapping and subcloning, thus, I
needed TONS of lambda DNA. I think I can honestly say I tried every
method out there: Maniatis, Red Book, Clontech, Wizard... The best luck I
had was using Promega's Lambda Sorb (it's simply an antibody to lambda, I
think) with liquid lysates: great yield, digested well, pure, etc. HOWEVER,
I found that it is possible for Lambda DashII to SCALE DOWN the amount of
Lambda Sorb 10-fold!! (a great weapon at budget meetings). I think it was
standardized for lambda gt10 which grows MUCH better than Lambda Dash II
(remember lambda genetics: the larger insert it can take, the more mutations
needed: lambda dash II (holds up to 23kb, I think) has 2 nin mutations
involved in the LYTIC cycle -- it's growth is therefore affected)
TO TEST THE LAMBDA-SORB EFFICIENCY (ie/ if you can scale down) I
recommend testing your liquid lysis supernatant AFTER the
incubation/precipitation/centrifugation of the LambdaSorb+lambda this way:
20 microliters lysis supernatant
2 microliters 2% SDS
2 microliters 0.1 M EDTA
incubate 5 minutes at room temperature--> run on a 0.7% agarose gel
You should NOT see a smear/band at the expected lambda kb (ie/ 9-20kb).
If you DO see a band, you need to add more lamdaSorb and re-precipitate
your leftover lambda. NOTE: You WILL see bacterial genomic DNA and RNA
(from lysed cells) so expect to see that.
Best of luck! I sympathize with all of you lambda-preppers!!
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