I normally synthesise my cDNA from RNA using Invitrogen's SuperscriptII
reverse transcriptase, and when done I store it at -20oC. I just make
dilutions from that stock for semiquantitative PCR and it generally
works well, and I can go back to the stocks months later and it's still
okay.

Last November (2 months ago) I prepared a batch of cDNAs, calibrated
them (the dilutions), did a bunch of PCRs, and this time I stored it at
-80, together with the RNA. No particular reason.
A month later I went back to use some of it, and failed. Then Xmas came
and I didn't try again till today... yep, it definitely does not work
anymore.

I think the fact I stored them at -80 and failed is a coincidence...
but I *never* have had this sort of problem before so I'm trying to be
open minded...

Has anybody experienced any problems with cDNA storage at -80?

I wonder if freeze-thawing is more harmful at -80 than -20... yet I
haven't experienced any problems at all with several rounds of
freeze-thawing at -20 in the past...

hmmm...

Greetings netters
I am trying to find a way to quantify relative expression of heat shock proteins
in seeds treated with different conditions. Does anyone have an idea for some
general antibody(s) that will cover as wide range of HSPs as possible?
Many thanks
Yoram

gerchman at research.haifa.ac.il

Hi,

Has anyone done any immunofluorescence against PIP2? If so, how did you
fix the cells?

I have a protocol involving a three-hour formaldehyde/glutaraldehyde
double fixation on ice, but if it was possible to do it at room
temperature or 37 C, at least to begin with, that would be a lot better
for the cells.

Thanks,
tom

Check your PCR DNTPs and prepare a new batch. Hopefully it helps!!
bye
Fernando Ariza

----- Mensaje original -----
De: Anajli Thomas gmail.com>
Fecha: Lunes, Enero 15, 2007 8:11 am
Asunto: RAPD breakdown

Hi all,

Is there any sonicator for bacteria cells disruption (20-100ml of
bacteria suspension to sonicate) that might (upon exchange of the tip)
be also used for much smaler volumes, 1 ml or less. I need to buy a
sonicator for bacteria and thought of getting sth versatile, suitable
also for homogenisation of small amounts of tissue (~0,5 ml). Of caurse
highly adjustable frequency and power would be required.

Thanks in advance for any ideas.

Lechu

Dear experts,

I hope I do not have a problem:

Intending to detect biotin derivatized proteins on PVDF membranes, I
have electroblotted my sds gel on the membrane and incubate it with the
biotin reagent o/n now. The next step would be blocking the membrane as
i usually do for western blotting with a buffer containg BSA.

I suddenly started worrying if BSA (0.5%%, together with 0.02%% TX100 in
buffer) is really suitable for this as I am afraid (fears are supported
by my beloved wife who is an expert, too (fortunately, of course ;-)
that I might introduce lotsa biotin and get strong background. My
worries are enlarged by a reference where the authors used "low biotin
containing BSA" from Aldrich, which I however cannot find in the
sial.com online catalogue. The original protocol I am cooking refers to
a kit with an undisclosed blocking agent.

As I do not want to waste my samples, I am asking you for urgent help.
My BSA is ordinary fraction V. Do I need to worry? What could I use
instead? On the shelf-stuff is preferred (ordinary gelatin is
available). Is there a way to kill biotin present in BSA by chemistry -
oxidation of the sulfur with H2O2 maybe?

Hi. I want to use Chelex-100 (Sigma Corp.) for extraction of DNA after using liquid nitrogen. I don't know exactly what to do and how to use it. I think it needs continuous mixing and sterilization is very importrant.
If someone could help me by giving me more details about this would help me very much. Thank you!