Problem with lambda GT10

Martyn K. White mkw6d at krebs.acc.Virginia.EDU
Mon Nov 12 19:01:39 EST 1990


To: GRAZIAWD at snyplava.bitnet
Subject: Re: Problem Query
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <9011121617.AA05492 at genbank.bio.net>
Organization: University of Virginia
Cc: 
Bcc: 

In article <9011121617.AA05492 at genbank.bio.net> you write:
>Date sent:  12-NOV-1990 11:01:52
>
>Recently we were having problems searching for an interferon regulatory
>protein clone in a lambda gt10 library. So, we decided to access the
>quality of our library by screening for bovine actin cDNA (SP64-actin).
>The plaques were unusual, i.e., quite small. So we decided to extract
>DNA from the library using the procedure in Current Protocols in Mol. Bio.
>Vol 1 (1.13.7) and hybridize the DNA blot (DNA -/+ EcoR1) to [32]P SP64-
>actin. Results showed the majority of signal at 2kb (insert is 2kb) with 3
>larger bands of much less intensity.However, one these signals was at ca. 50kb
>for both -/+ EcoR1. Surprise and confused! Since the procedure requires the
>use of DNase & RNase treatment to lysate prior to phenol extraction, could
>this be the problem. Any suggestions or help would be appreciated. We are
>in the process of extracting the DNA without DNase & RNase. Send responses
>to GRAZIAWD at SNYPLAVA.BITNET. Thanks in advance.
>
>Regards,
>        Bill :-)
>
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I think the problem is that in libraries in lambda GT10, one or both
of the EcoR1 sites flanking the insert can be lost by some unknown 
mechanism.  This occurs at a surprisingly high rate and is very
annoying.  Thus I have isolated clones and then tried to subclone
them using EcoR1 only to find that in a significant percentage of the
clones EcoRI will not cut at either one or both sites. This is due
to a mutation in the sequence and not to "dirty DNA". 
Thus your three extra bands would be:
	actin + short arm of GT10 (1 EcoR1 site lost)
	actin + long arm of GT10  (other site lost)
	actin + both arms  (both EcoR1 sites lost)

You could test this (if you are interested) by cutting with both
HindIII and BglII which should give you a single band which is
somewhat bigger than the expected size of the insert (if my theory
is correct).  HindIII and BglII flank the "EcoR1" sites.

Clones with this problem can be subcloned using these two restriction
enzymes into suitable polylinker-containing plasmid and sequenced at
their termini using lambda GT10 primers (BRL).


 
-- 
	Martyn K. White, Box 441, U.Va, Cville VA 22908
	     		(804) 924 8710	
                 mkw6d at krebs.acc.Virginia.EDU
		      mkw6d at Virginia.EDU

This is my .signature and I will say what I like.
	Martyn K. White, Box 441, U.Va, Cville VA 22908
	     		(804) 924 8710	
                 mkw6d at krebs.acc.Virginia.EDU
		      mkw6d at Virginia.EDU



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