bionet.molbio.methds-reagnts Google Group

Re: problem in immunoprecipitation experiment

engelbert_buxb...@hotmail.com (Dr Engelbert Buxbaum) — Wed, 19 Jun 2013 17:36:39 UT

In article <mailman.445.1370726150.10461. meth...@net.bio.net>,
divya.med...@gmail.com says...

Not necessarily related to your problem, but try formalin-fixed Staph.
aureus cells (e.g., Pansorbin) instead of Protein A beads. A lot
cheaper, and in my hands binding is better.

Rhizobium Genomic DNA Extraction

carito2...@gmail.com (Carolina Mazo) — Fri, 14 Jun 2013 20:35:46 UT

Good day Gerd Nilsen,

My name is Carolina Mazo, I am a Colombian microbiologist working with
Rhizobium specifically with 16S rRNA pcr. I just found your question at
[link] and I was wondering if you could send me your DNA extraction
protocol for rhizobium

Thank you very much.

--
Carolina Mazo

Re: Mysterious plasmid in E.coli affecting cell growth

d...@no.email.thankstospam.net (DK) — Fri, 14 Jun 2013 03:01:25 UT

Indeed! Thanks for clueing me in!

Re: Mysterious plasmid in E.coli affecting cell growth

pj...@cam.ac.uk (Peter Ellis) — Thu, 13 Jun 2013 06:53:51 UT

... don't they all? The Blue in the name is there precisely because you
can do blue/white selection: i.e. there's a lac operator and peptide there!

Peter

RE: Mysterious plasmid in E.coli affecting cell growth

d...@no.email.thankstospam.net (DK) — Thu, 13 Jun 2013 03:24:25 UT

Thanks!

I did not know that some Bluescript plasmids have lac operator.
The inserts in our case are cloned under T7 promoter and thus
lac should be in the opposite orientation. But our inserts can
very well be toxic (ion channels) and the plasmid is likely to have
long and tortured history, so I would not discount a possibility

RE: Mysterious plasmid in E.coli affecting cell growth

alejandro.mar...@cigb.edu.cu (Alejandro Miguel Martin Dunn) — Wed, 12 Jun 2013 15:00:28 UT

Perhaps you could isolate the problem by trying an F- strain containing the lacIq allele. The sole role of the F' episome in TOP10 F' (as in XL-1Blue) is to provide a lacIq gene. If your plasmid is a pBluescript/pUC19 derivative it might still have a lac promoter, which would then not be fully repressed in TOP10 or E.cloni 10G, and if your insert is toxic somehow...

Re: problem in immunoprecipitation experiment

pj...@cam.ac.uk (Peter Ellis) — Tue, 11 Jun 2013 20:02:15 UT

Presumably a typo for IGEPAL. Very common detergent, it's the
replacement for NP-40 (which is no longer produced).

so 2000x more dilute. Could you use more protein?

He said purified protein, not cell lysate. If he's using 1/2000 the
concentration relative to your protocol, it'll be equivalent to a

Re: problem in immunoprecipitation experiment

irapp...@scripps.edu (Irit Rappley) — Mon, 10 Jun 2013 18:12:34 UT

Hi Divya,

A few questions for you:
1. What is IEPAL?
2. Why do you use only 1ug protein and only 15ul beads in 2mL IP volume? It seems too dilute to me. I usually IP from a 1 mg/mL solution of cell lysate; yours is 0.5 ug/mL, so 2000x more dilute. Could you use more protein?
3. Could you please provide more details about your IP protocol? Is your antibody pre-conjugated to the beads, or do you add the antibody and the beads separately? If so, how much antibody are you adding? What is the order of steps that you perform? How long are your incubations with the antibody / beads? How are you eluting your protein off the antibody?

problem in immunoprecipitation experiment

divya.med...@gmail.com (divya teja) — Sat, 08 Jun 2013 20:09:39 UT

Hello everyone,

Iam doing immunoprecipitaion experiment with Protein A/G beads(15ul) in PBS
buffer Ph 7.4 of volume 2ml. Iam taking 1microgram of my purified protein.
washing beads 4 times with 0.25% IEPAL.

My problem is my purified protein is going and binding to the beads
unspecifically.

Re: benzoylation of polyamines

engelbert_buxb...@hotmail.com (Dr Engelbert Buxbaum) — Fri, 07 Jun 2013 16:08:00 UT

In article <mailman.436.1370538219.10461. meth...@net.bio.net>,
campane...@mail.montclair.edu says...

I am not familiar with the specific method you mention, but some general
advice may be in order:

- How fast would benzoyl chloride react with your mobile system? Partial
hydrolysis could explain multiple peaks.

benzoylation of polyamines

campane...@mail.montclair.edu (James J. Campanella) — Thu, 06 Jun 2013 16:55:44 UT

I don’t know if anyone out there has performed this
protocol, but I am looking for advice. We are trying to determine the
concentration of

polyamines in some plant tissue samples using a
benzoyl-labelling process and HPLC. Unfortunately, we have run into some
problems and

not even gotten beyond the first step of labeling standards.

Problems extracting DNA with magnetic beads

n.thoms...@massey.ac.nz (Thomson, Neroli) — Tue, 04 Jun 2013 00:26:54 UT

Hello all,

Apologies if you receive this twice, my personal email is playing up, so I’ve sent it from my work address.
I’m having afew problems with DNA extraction and was hoping someone out there may have some suggestions.
As part of my PhD, I am running a qPCR looking for a papillomavirus (small double stranded DNA virus) in feline skin. I have collected just under 400 samples of plucked hairs and skin swabs from clinically normal cats, including 1 day old kittens, older kittens and adults. The samples are in 100µL 0.9%NaCl at -20⁰C (I’m only looking for DNA not RNA).

Why to use N-lauroylsarcosine for Hybridization?

michel.sar...@hotmail.com (michel saremi) — Thu, 02 May 2013 15:46:18 UT

Did you get an answer?
I am asking the same question.

In the protocols I have found, it has been used as a prehybridization step, along with formamide.
The molecule is pretty big, I suspect it is used to sandwich itself between the two strands of a DNA or RNA, while the formamide deionizes the the bases, stabilizing dehybridization, prior to Probe-hybridization, but I am guessing.

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