Methods Digest, Vol 3, Issue 4

TieQiao Wu TieQiao.Wu at baker.edu.au
Thu Aug 4 20:03:35 EST 2005


Hi, everyone:

According to my experience to handle the Bio-Rad protean 2 system, I check the edges of glass plates carefully and discard those plates with seriously damaged edges. After using of glass plates I always use hot tape water to wash plates thoroughly. At the moment, we use another system called Hoefer Mighty small -multiple gel caster from Amersham Pharmacia. Using this system, you can cast upto 14 or 15 gels each time. Amersham also has multiple gel transferring system.

Cheers

Tieqiao     

-----Original Message-----
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu]On Behalf Of
methods-request at oat.bio.indiana.edu
Sent: Friday, 5 August 2005 3:00 AM
To: methods at magpie.bio.indiana.edu
Subject: Methods Digest, Vol 3, Issue 4


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Today's Topics:

   1. suggestions for new PAGE apparatus (Dr. James J. Campanella)
   2. RT-PCR (Johanna Fehling)
   3. RE: suggestions for new PAGE apparatus (Jayakumar, R)


----------------------------------------------------------------------

Message: 1
Date: Wed, 03 Aug 2005 09:52:22 -0400
From: "Dr. James J. Campanella" <campanellj at mail.montclair.edu>
Subject: suggestions for new PAGE apparatus
To: methods at magpie.bio.indiana.edu
Message-ID: <6.1.0.6.0.20050803095118.03ab0db0 at mail.montclair.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Hi everyone,

I am in the market to buy a new PAGE set-up. I refuse to buy Biorad again. 
I have been burnt by their low quality equipment before. We have begun 
pouring a large number of gels, and  I am sick of wasting half my time time 
trying to keep the casting system from leaking even as I'm pouring my gel. 
There has to be something better out there than Biorad. I would like 
recommendations on which company's apparatus will allow me to pour my PAGEs 
every time without leakage.

Thanks for the help,

Jim

James J. Campanella,
Associate Professor
Department of Biology and Molecular Biology
Montclair State University
1 Normal Avenue
Montclair, NJ 07043

Alternate email address: jcamp at alumni.uchicago.edu

Ph: 973-655-4097
Fax: 419-791-9834 



------------------------------

Message: 2
Date: Wed, 3 Aug 2005 16:41:16 +0100
From: Johanna Fehling <jf510 at york.ac.uk>
Subject: RT-PCR
To: methods at magpie.bio.indiana.edu
Message-ID: <78792533230d52b2bd7eb5cc39920ff3 at york.ac.uk>
Content-Type: text/plain; charset=US-ASCII; format=flowed

Does anyone know of an RT-PCR kit or protocol that would select for 
long polyA tails? I am trying to do RT-PCR on protozoan cell cultures 
contaminated with a mix of bacteria. Getting rid of the bacteria 
doesn't seem to be an option (many things have been tried). Although 
some bacterial transcripts are polyA'd, I think these tend to be much 
shorter than in euk transcripts. I've been using the Strategene kit 
"StrataScript Two-tube RT-PCR system with PfuUltra" but still seem to 
be getting a lot of bacterial genes in my PCR products. I know polyU is 
supposed to be more selective than polydT. Does anyone know of any kits 
that use polyU, or any other tricks I might try?

Cheers,
Johanna

--------------------------------------------------------
Johanna Fehling
Postdoctoral Research Fellow
Department of Biology, Area 3
University of York
PO Box 373
York, YO10 5YW
United Kingdom

phone#: ++44 (0)1904 328633
fax#: ++44 (0)1904 328505
--------------------------------------------------------



------------------------------

Message: 3
Date: Wed, 3 Aug 2005 17:53:37 -0400
From: "Jayakumar, R" <R.Jayakumar at roswellpark.org>
Subject: RE: suggestions for new PAGE apparatus
To: "Dr. James J. Campanella" <campanellj at mail.montclair.edu>,
	<methods at magpie.bio.indiana.edu>
Message-ID:
	<97101976F8A044468CA74FE11883B90E07496514 at VISTA.roswellpark.org>
Content-Type: text/plain;	charset="us-ascii"


Hi.. I can see a lot of frustration directed at BioRad here.. :-))  I
too had problems earlier with Protean 2.  After much trials and carefull
observation I found the problem and after that the plates have probably
leaked only 2-3 times in the past 3 years.  The leak is most often due
to the following reasons.  This is with my experience using mini protean
2 as well as the larger protein xi. 
  1.  The bottom of the spacers don't exactly align with the plate edges
(run your finger through it check whether the spacer is flush with the
glass edges).  Gel solutions can diffuse out through these spaces.
  2.  Glass plates often develop very small nicks and crack at the edges
due to use or due knocking against each other while washing.  The gel
solution seeps out through these tiny nicks.  Run a finger over the
edges to ensure that the edges are perfectly smooth. 
  3. Also check whether there is any solidified acrylamide sticking
between the spacers (to avoid this, clean and flush the casting
apparatus thoroughly under the tap IMMEDIATELY after use).  Solidified
pieces of acrylamide can easily protrude between the spacers and the
glass causing the solution to leak.
   4. These solidified pieces of acrylamide can also cause the glass
plates to crack when the screws are tightened.  I have cut down on glass
breakage by more than 2/3rd by following a strict washing routine.  
  These will ensure a sure leak proof system and many more years of
happy casting experiences. 
   5. Also if the gel casting holders don't snap into place, change the
red foam pads (you can buy them separately from Biorad and stick them
into place). They get crushed with repeated use.  Alternatively you can
try placing pads of whatman no3 paper between the red foam pad and the
grey cushion. 

Best of luck
Jai

PS>  I haven't found a better SDS-PAGE apparatus yet than bio-rad.  

-----Original Message-----
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Dr. James J.
Campanella
Sent: Wednesday, August 03, 2005 9:52 AM
To: methods at magpie.bio.indiana.edu
Subject: suggestions for new PAGE apparatus
Importance: High

Hi everyone,

I am in the market to buy a new PAGE set-up. I refuse to buy Biorad
again. 
I have been burnt by their low quality equipment before. We have begun 
pouring a large number of gels, and  I am sick of wasting half my time
time 
trying to keep the casting system from leaking even as I'm pouring my
gel. 
There has to be something better out there than Biorad. I would like 
recommendations on which company's apparatus will allow me to pour my
PAGEs 
every time without leakage.

Thanks for the help,

Jim

James J. Campanella,
Associate Professor
Department of Biology and Molecular Biology
Montclair State University
1 Normal Avenue
Montclair, NJ 07043

Alternate email address: jcamp at alumni.uchicago.edu

Ph: 973-655-4097
Fax: 419-791-9834 

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