Re: [ccp4bb] Shredded E coli pellets

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Shredded E coli pellets
From: "Mark J {- dot -} van Raaij" mark {- dot -} vanraaij {- at -} USC {- dot -} ES
Date: 2009-07-02

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Subject: Re: Shredded E coli pellets
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-07-02

Subject: Re: Shredded E coli pellets
From: Paul Leonard leonard {- at -} CABM {- dot -} RUTGERS {- dot -} EDU
Date: 2009-07-02

Dear Jacob,

I believe this paper will really help you out (see attached pdf).

Marcin Los, Agata CZY, Eugenia SELL, Alicja WÊGRZYN, Peter NEUBAUER,
Grzegorz WÊGRZYN
J. Appl. Genet. 45(1), 2004, pp. 111-120
Bacteriophage contamination: is there a simple method to reduce its
deleterious effects in laboratory cultures and biotechnological factories?

As well as the method suggested in the paper I recommend you prepare new
glycerol stocks from freshly transformed colony (using cells and equipment
that you are certain are not contaminated).

Finally you may wish to consider using T1 phage resistant strains of Ecoli
for future work.

Sigma and NEB sell BL21 (DE3) T1R (T1R = T1 phage resistant) cells.
I think Invitrogen cell strains for cloning and DNA manipulation that are
T1 phage resistant.
There may well be other strains and suppliers I am not aware of.

Good luck.

Paul

> Okay, it seems that the consensus is phage infection. Is there anything to
> seal the diagnosis? Also, does anybody have literature on de-phaging
> glassware? I am assuming that regular autoclaving will not do the trick?
>
> Jacob
>
> *******************************************
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> Dallos Laboratory
> F. Searle 1-240
> 2240 Campus Drive
> Evanston IL 60208
> lab: 847.491.2438
> cel: 773.608.9185
> email: j-keller2@northwestern.edu
> *******************************************
>
> ----- Original Message -----
> From: "Jacob Keller"
> To:
> Sent: Thursday, July 02, 2009 11:42 AM
> Subject: [ccp4bb] Shredded E coli pellets
>
>
>> Dear crystallographers,
>>
>> I recently expressed some new constructs, and found after my usual
>> expression protocol that the cell pellets were not compacted at the
>> bottom
>> corner of the bottles us usual, but were instead smeared as a film on
>> the
>> side, and further, were somewhat clumpy, like clots, and with a smaller
>> pellet in the usual location. The centrifugation was exactly as usual. I
>> noticed that there was also a bit more foam in the medium than usual,
>> but
>> I am not convinced that this was the issue, although it might be a
>> symptom. My suspicion is that the constructs are lethal and cause cell
>> lysis, but I am not sure. Has anybody seen this phenomenon before, and
>> gotten to the bottom of it?
>>
>> Jacob Keller
>>
>> *******************************************
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> Dallos Laboratory
>> F. Searle 1-240
>> 2240 Campus Drive
>> Evanston IL 60208
>> lab: 847.491.2438
>> cel: 773.608.9185
>> email: j-keller2@northwestern.edu
>> *******************************************
>>
>

Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leonard@cabm.rutgers.edu

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Shredded E coli pellets
From: "Mark J {- dot -} van Raaij" mark {- dot -} vanraaij {- at -} USC {- dot -} ES
Date: 2009-07-02

Next message:
Subject: Re: Shredded E coli pellets
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-07-02