Large plaques in lambda libraries

CLONTECH at BIOTECHNET.COM CLONTECH at BIOTECHNET.COM
Wed Jan 19 18:50:39 EST 1994


In article <9309181831.AA08215 at rockvax.ROCKEFELLER.EDU> posted 
on 18 Sep. 1993, Diana Horvath at ralston at ROCKVAX.ROCKEFELLER.EDU 
writes:
>
>I have recently purchased a tobacco genomic library, which
>when plated gives rise to 1-5 large plaques per 50,000
>lambda plaques.  These large plaques are evident hours
>before (>6 h) the lambda plaques and grow in size to 4-5 mm
>by 9 hours.
>
>Others in our lab suggest that these may be T phage and that
>T phage get absolutely everywhere.  So, ...

This is CLONTECH's response:

Presence of large plaques in CLONTECH's lambda libraries

We have detected the presence of larger than normal size plaques 
in several premade lambda libraries. The large plaques are 3-4 
times larger than the plaques normally formed by lambda phage, 
and the large plaques are apparent after incubation for only 
5-6 hours at 37C (normal lambda plaques are apparent after 8-9 
hours of incubation at 37C).  The frequency of the large plaques 
varies from 1-5 per 100,000 normal plaques.

The morphology and the size of the large plaques appeared to be 
those formed by the T-series phage. Cross contamination is 
unlikely because reagents, equipment, and disposable containers 
are dedicated to library construction. All containers are used 
only once, and UV irradiation is used for decontamination. Even 
though the large plaques have not been observed in all lambda 
libraries, we decided to investigate each component used in 
library construction. Those components included restriction 
enzymes, modifying enzymes, buffers, dNTPS, control mRNA, vector 
DNA, and in vitro packaging extract.

The least-suspected component, in vitro packaging extract 
(purchased from a leading supplier), was found to yield about 30 
morphologically similar large plaques when packaging extract alone 
was plated on a bacterial lawn and incubated for 5P6 hours at 
37C. (Note that the frequency might be higher in somecDNA libraries 
packaged using the extract because more extract must be used to 
obtain a representative library.)

A new packaging extract from a different supplier, Amersham, was 
thoroughly tested. We observed no large plaques; therefore, the new 
packaging extract was used to construct lambda libraries to replace 
all the libraries known to contain the large plaque contamination.


Sailaja Kuchibhatla
CLONTECH Laboratories, Inc.



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