Question: lambda plaques not transparent, bacterial lawn not opaque

RPA Groot rpa.groot.oncol at med.vu.nl
Mon Jan 29 11:02:37 EST 1996


In article <DLp4L5.4o7 at uns.bris.ac.uk>, Harry.Witchel at bristol.ac.uk (Harry J. Witchel) says:
>
>Hello Fierce Molecular Wizards --
>        I have an amplified library from Stratagene, and I suspect I am not 
>growing it correctly.  The library is in lambda zap II, the host is XL1 blue 
>MRF' as recommended, and I am growing it at 37C on LB agar with LB top 
>agarose.  Two unexpected things have occurred.  
>
>        1)  The plaques are frosty rather than clear (translucent rather than 
>transparent), but the plaques are uniform in size and are frosty throughout, 
>such that the bacterial lawn is just a bit thinner than elsewhere.
>
>        2)  The bacterial lawn is not opaque (even when the bugs are not 
>infected).  There is definitely a bacterial lawn, but the bugs have not grown 
>nearly as thick and as opaque as they were when I grew the same bugs as 
>colonies.
>
>Note that I have exactly the titer Stratagene claim.  I think I have followed 
>all the Stratagene protocols to the letter except for the following:
>
>I have used LB instead of NZYM for my plates
>My plates are a week old (instead of two days old)
>I pour my top agarose at 52C instead of 48C (otherwise the agarose hardens too 
>quickly)
>I use ~10 ml of top agarose per 150 mm plate (they recommend 8 ml)
>I dilute my phage in lambda diluent rather than SM
>I grow my bugs overnight and dilute to approximately the right density (they 
>recommend a log phase set of bugs and to dilute exactly using a 
>spectrophotometer)
>
>Can anyone tell me which of these steps are critical in the procedure and 
>which can be varied.
>        Thanks in advance, and clone wisely,
>        Harry
>
>Harry.Witchel at Bristol.Ac.Uk
>


Hi,


Your methods are fine.
Just use a differnt host strain; VCS 257 (Stratagene) or JM 105 (Pharmacia)


Richard



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