Large plaques in lambda libraries

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Jan 27 17:04:36 EST 1994


--------------------------------------------------------------------------------
Most of you are probably aware of the thread of discussions that took place a
few months ago concerning the contamination of commercial genomic libraries
with T phages. Very recently, an article about this issue was published in the
December 1993 issue of TIBS:

@article{Hengen1993Dectibs,
author = "P. N. Hengen",
title = "Methods and Reagents: Hybridization doughnuts and
uninvited phages",
journal = "Trends in Biochemical Sciences",  
volume = "18", 
number = "12",
pages = "484-485",
month = "December",    
year = "1993"}

I am not exactly sure how much influence this article had on the quick response
of CLONTECH in this matter, but I would like to say that I am very impressed
with the way in which they have handled the problem. I have no doubt that this
forum is reaching the commercial suppliers for improved quality of products and
services, and that our collective voices are being heard.

Thank You CLONTECH for providing this for us.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************

[CLONTECH's solution follows]
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In article <2hkh0f$hfb at mserv1.dl.ac.uk> CLONTECH at BIOTECHNET.COM writes:

> 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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