LIBRARY PLATING: no lysis of bacterial lawn

Angela Jones A.C.Jones at bham.ac.uk
Thu Apr 7 09:27:56 EST 1994


In article <2nve5n$mpq at gazette.bcm.tmc.edu>,
khwang at SPECIALK.IAIMS.BCM.TMC.EDU (Kevin O Hwang) wrote:

> HELP!
> We are attempting to plate out two libraries, one is a lambda ZAP and
> the other is lambda gt10. The host cells for the ZAP library are from
> an LB plate and are XL1 blues. The gt10 host cells are C600 hfl, and
> are from a frozen stock, streaked out on a plate and a single colony
> chosen. The cells are both grown overnight in 10 mM MgSO4 and 0.2% maltose.
> They are spun down, resuspended in 10 mM MgSO4 and used at an OD of .5
> The libraries are diluted in SM and plated out on fresh, dry NZY plates
> following incubation of the bacteria and phage for 15 min at 37C. The
> resulting lawn is PERFECT but, for the third time, there is NO LYSIS,
> no clearing, nothing!!!! Does anyone have any ideas? I've screened over
> 20 million phage previously, and the library (at least one of them)
> is known to be good (as of 6 months ago, and it's been stored at 4C since
> that time).
> 
> Many thanks in advance
> Kevin and Jill

Hi there,
        I had the same problem plating a lamda ZAP library on XL1-blue a
few 
months ago. I got over the problem by using a less rich medium such as TB:
10g bacto tryptone; 5g NaCl; 11g agar (hard) / 7g (soft); 10 ml 1M MgSO4.
I grew the cells overnight in TB broth + 10mM MgSO4 + 0.2% maltose at 30C;
the cells stop growing at an OD600 of 0.5-0.6. They are spun down,
resuspended in 10mM MgSO4 and used at an OD of 0.5. The phage are diluted
in lamda diluent. Both the top and bottom layers of agar are TB medium. I
had been using your protocol successfully until recently when I changed
labs - I think the things are quite fussy. Good luck!



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