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Han | 1 Apr 2010 03:27
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Re: Help!! Membrane isolation from blood platelets

Tina Hofmaier <tina.hofmaier@...> wrote in
news:mailman.377.1270072792.25217.methods@...: 

> Hi,
> is there anyone who has  experience with blood platelets. I did a
> step-by-step centrifugation protocol, but I get too low protein at the
> end. 
> 
> Thanks a lot
> 
> 
> tina

M. J. Broekman, N. P. Westmoreland, and P. Cohen. An improved method for 
isolating alpha granules and mitochondria from human platelets.  J.Cell 
Biol. 60:507-519, 1974.

I am at mjbroek at med dot cornell dot edu

--

-- 
Best regards
Han 
email address is invalid
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Salama Ouf | 5 Apr 2010 00:02
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antibodies of dermatophytes

Dear Stefen
I would be appreciating providing me with any report (s) and  published papers about antibodies
specific for cutaneous diseases.
 
Best wishes
 
Dr. Salama A. Ouf 
Professor of Microbiology 
Department of Biology, 
Faculty of Science, Taibah University 
Almadinah Almunawwara, P.O. 30002 
Saudi Arabia 
e-mail: saoufeg@... 
Tel (Mobile) : +966 569 184508 
       (Home):   +966 4828 4508
Fax: +966 48454770
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LeboMad | 6 Apr 2010 11:02
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PCR failing

Hi all. I have a serious problem with PCR amplification in my lab. I
have done all troubleshooting there is out there I can't seem to get
it right. I normally get nice clear bands but for some reason I get
very faint bands if any at all. Can anyone suggest anything. I've got
a student that needs some PCR runs and needs result as in yesterday.
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Christian Praetorius | 6 Apr 2010 15:05
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Re: PCR failing

LeboMad <lmadubanya@...> wrote:

>have done all troubleshooting there is out there I can't seem to get
>it right. I normally get nice clear bands but for some reason I get
>very faint bands if any at all. Can anyone suggest anything. I've got
>a student that needs some PCR runs and needs result as in yesterday.

The nyou should describing *exactly* what your procedure is and what
you tried to troubleshoot.

Christian

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-- 
X-no-Sig: yes
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Cristina Echevarría Ruiz de Vargas | 6 Apr 2010 15:44
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protease cleavage site program

Dear colegue,
I has sow your e-mail direction associated with a program to evaluated possibles cleavege sites in a
protein. I have try to use this information but it was not possible probably because  I dont know. So I will
send you a peptide ERHHSIDAQLRALAPGKVSEE  and I would ask you the possibility to look a possible cleavege
site in this peptides. Sorry for my Inglish. A would  apreciated very much if you can loock for this as I am
working with a protease that seem to attack this peptide.

Tahnk you very much in advance

Cristina Echevarría

*****************************************************
Cristina Echevarría
Profesora Titular  de Universidad
Dpto. Biología Vegetla y Ecología 
Facultad de Biología 
Universidad de Sevilla
Sevilla
España
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Sébastien Vigneau | 6 Apr 2010 16:48
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Re: PCR failing

Hi,

If the PCR used to work but is not working anymore, you may have some PCR
inhibitors in your samples. Diluting them in water, about 100 times, can
help (but trying different dilution factors, e.g. 10, 100 and 500 may be a
good idea). Alternatively, your template concentration may be too low or
your DNA may be degraded.

If this is a new PCR, you may lower the annealing temperature or increase
the concentration of primers (generally in the 0.2 µM - 0.8 µM range for the
final concentration). However, in the first case, you may loose specificity
and in the second, you may generate primer dimers.

I hope this helps.

Sébastien

On Tue, Apr 6, 2010 at 9:05 AM, Christian Praetorius <prae@...> wrote:

> LeboMad <lmadubanya@...> wrote:
>
> >have done all troubleshooting there is out there I can't seem to get
> >it right. I normally get nice clear bands but for some reason I get
> >very faint bands if any at all. Can anyone suggest anything. I've got
> >a student that needs some PCR runs and needs result as in yesterday.
>
> The nyou should describing *exactly* what your procedure is and what
> you tried to troubleshoot.
>
> Christian
(Continue reading)

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StewJW | 6 Apr 2010 17:33
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Re: MTT Cell Viability Assay

Here's an alternative XTT method in 96 well microplate format we used
in our lab. XTT has a higher sensitivity and a higher dynamic range:

Cytotoxicity was measured the following day by the addition of 80 μl/
well XTT/Phenazine methosulfate (PMS) solution (XTT solution, 2 mM in
PBS, pH 7.4, pH
adjusted to 6.0-6.5; PMS solution, 0.92 mg/ml in PBS, pH 7.4;
solutions mixed at a ratio of 8
ml XTT solution to 200 μl PMS solution) followed by an incubation time
of 1.5h at 37°C.
Absorbance was read at 490 nm with scatter correction at 690 nm.

But check the literature.
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Yoram Gerchman | 7 Apr 2010 08:04
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Re: protease cleavage site program


Dear Cristina
Expasy <www.Expasy.ch> is a great site for DNA, RNA and protein related tools.
For your specific question try <http://www.expasy.ch/tools/peptidecutter/>.
Yoram

>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 6 Apr 2010 02:02:42 -0700 (PDT)
> From: LeboMad <lmadubanya@...>
> Subject: PCR failing
> To: methods@...
> Message-ID:
> 	<5a4d0356-fa5d-45ea-8b98-4b1eed90bc72@...>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi all. I have a serious problem with PCR amplification in my lab. I
> have done all troubleshooting there is out there I can't seem to get
> it right. I normally get nice clear bands but for some reason I get
> very faint bands if any at all. Can anyone suggest anything. I've got
> a student that needs some PCR runs and needs result as in yesterday.
>
>
> ------------------------------
>
> Message: 2
> Date: Tue, 06 Apr 2010 13:05:32 +0000
> From: Christian Praetorius <prae@...>
(Continue reading)

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DK | 7 Apr 2010 14:58

Re: PCR failing

In article
<5a4d0356-fa5d-45ea-8b98-4b1eed90bc72@...>,
LeboMad <lmadubanya@...> wrote:
>Hi all. I have a serious problem with PCR amplification in my lab. I
>have done all troubleshooting there is out there I can't seem to get
>it right. I normally get nice clear bands but for some reason I get
>very faint bands if any at all. Can anyone suggest anything. I've got
>a student that needs some PCR runs and needs result as in yesterday.

Imagine a student coming and telling you that "nothing works, please
help me" - without any pertinent details. Would you be able to help?

DK
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Alex - Exon Biotecnologia | 7 Apr 2010 18:57
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Long Ranger Gel - Bad polymerization

Hi all,

I am used to prepare Long Ranger gel solutions to run DNA small fragments
(microsatellites) in a ABI 377 sequencer.

Since the beggining of the week, I have had problems with the polymerization
of the gel even using the same reagents I used before. We tried to change
all the reagents, but this didn't work. Normally, we wait 1:30 hour to load
the gel and start the run.

Does anybody know what might be the problem? It's very cold here since
sunday, but I don't think this can be the reason of the problem...

Thanks,

Alex
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Gmane