gmane.science.biology.methods-reagents

Why to use N-lauroylsarcosine for Hybridization?

michel saremi — 2013-05-02T15:46:18

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.

I look forward to hear your thoughts on the matter.

Best Regards 

Michel Saremi

Re: RNA/Protein UV crosslinking (UVXL) assay

DK — 2013-04-27T04:12:54

0.2% detergent will lyse sizeable fraction of plasma and ER-derived 
membranes. Being low salt, it will cause some F-actin depolymerization. 
And of course the degree of cytoskeletal fraction contamination will 
critically depend on how the lysate is spun. 16 Kg in eppy vs 300 Kg 
in ultra can make all the difference in the world. Finally, "freshly 
added protease inhibitors" is pretty meaningless without specifying 
what they are and at what concentration (and, in practice, how they 
were stored before being "freshly added"). 


Nuclei - definitely not, mitochondia - almost none, everything else -
definitely yes but to a varying degree (and also depends on cell 
concentration). 


With detergent present, there shouldn't be much difference but your 
homogenization is the best. 


I'd try: permeabilize cells and wash to remove bulk of cytosolic 
proteins (check out a simple freeze-thaw way of doing it: 
http://www.ncbi.nlm.nih.gov/pubmed/9790867 ), then homogenize
the hell out of them, spin down nuclei at lo

Re: Any experiences with Geneamp 9700 cycler?

Michael Sullivan — 2013-04-26T16:01:13

Yeah, I haven't quite figured out why they designed it this way, although the tube holder is sort of nice for working with the strip tubes. I don't think there's a way to adjust how much pressure the lid applies (like the MJ DNA engine [now biorad?] has), so maybe ABI's approach with the 9700 was to just apply a lot of pressure and have a device to keep the tubes from being crushed?

My experience with the 9700 was rather like yours, I think. When we first got it, I crushed a bunch of tubes, because who consults the manual for how to put tubes into a PCR machine? :)

I initially thought that since I was not using the ABI tubes, that was the problem. So once I bought the ABI tubes and crushed some of those, I realized there must be something wrong.

I think maybe the 2720 applies less pressure from the lid than the 9700.... at least that's how it feels to me, so that's probably why you were able to use it without crushing your tubes.

Anyway, I'm glad you have that problem fixed!

Mike
---
Michael L. Sullivan

RNA/Protein UV crosslinking (UVXL) assay

Peter Ellis — 2013-04-26T09:23:35

Dear all,

I am interested in an mRNA that has been described as being bound to the 
cytoskeleton.  There are two published papers showing that a labelled 
fragment of the 3' UTR can be crosslinked to various (unidentified) 
proteins.  I am hoping to identify the proteins and characterise them 
further.

Unfortunately I am having great difficulty in replicating the original 
findings.  In our hands the UVXL experiment only gives very low intensity 
signal, the band sizes are different to the published work (though I note 
that the two published papers also reported a range of band sizes!), and 
in our hands the binding cannot be competed by antisense oligos, so it's 
not a sequence-specific interaction).

The UVXL binding assay uses a protein extract made with a cytoplasmic 
extraction buffer (CEB).  Then, a labelled RNA probe (e.g. DIG or biotin 
labelled) is added, allowed to interact with the proteins in the presence 
of competitors (e.g. heparin, tRNA, other irrelevant unlabelled RNA). This 
binding step

Re: Any experiences with Geneamp 9700 cycler?

DK — 2013-04-26T03:39:25

Now that I've got the trays, I have no problems too. It just puzzles me
that while 2720 and 9700 formally list the same requirements for 
plasticware, 2720 is prefectly usable without any trays who 9700 
is not. 

Bad design, IMO. 

DK

Re: Plasmid mix in the clone - the mechanism?

DK — 2013-04-26T03:34:26

Yes, easily separated. 


Have never seen that in 10+ years. 

Those would show up in sequences as repeats (or near-repeats)
instead of what I see as mixtures. 

DK

Re: Plasmid mix in the clone - the mechanism?

Jonathan Rupp — 2013-04-25T17:01:22

Does this mean you can isolate both the new and parental plasmids from your
mixes?  If not, I suspect you have duplicate regions in a single plasmid.
We have seen this numerous times with quick change.  Not sure the
mechanism, but the result is often as if the primers were ligated end to
end.


On Wed, Apr 24, 2013 at 11:36 AM, Pow Joshi <pow.joshi-Re5JQEeQqe8AvxtiuMwx3w< at >public.gmane.org> wrote:

RE: Plasmid mix in the clone - the mechanism?

Dunowska, Magda — 2013-04-25T06:22:26

Here is a link to a discussion on the topic that I found useful at the time we seemed to have a similar problem (mix of plasmids from one plasmid prep): http://bitesizebio.com/articles/plasmid-retention/

Magda

________________________________________
From: methods-bounces-RhB9QHrfm5AZm++ksbtxhJVzexx5G7lz< at >public.gmane.org [methods-bounces-RhB9QHrfm5AZm++ksbtxhA< at >public.gmane.orgedu] on behalf of Pow Joshi [pow.joshi-Re5JQEeQqe8AvxtiuMwx3w< at >public.gmane.org]
Sent: Thursday, April 25, 2013 7:36 AM
To: Vladimir Gainullin
Cc: methods-ROGg2w78f1QLwOoBEzVeC1Bd07bTekyB< at >public.gmane.org; DK
Subject: Re: Plasmid mix in the clone - the mechanism?

On 24 April 2013 12:55, Vladimir Gainullin <gainullin-Re5JQEeQqe8AvxtiuMwx3w< at >public.gmane.org> wrote:

The possibility of concatamers? I understand that they are possible is
various situations of cloning.

Pow

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Re: Plasmid mix in the clone - the mechanism?

Pow Joshi — 2013-04-24T19:36:44


The possibility of concatamers? I understand that they are possible is
various situations of cloning.

Pow

Re: Plasmid mix in the clone - the mechanism?

Vladimir Gainullin — 2013-04-24T16:55:37

DpnI digest?


On Wed, Apr 24, 2013 at 12:39 AM, DK <dk-ExnEkaPBXRK421ATBNoTQIIY+NxbBFVqXqFh9Ls21Oc< at >public.gmane.org> wrote:

Plasmid mix in the clone - the mechanism?

DK — 2013-04-24T05:39:17

Wonder if anyone has good explanation as to how this is occuring: 

Once in a while, looking at sequences of mutant clones obtained by 
"QuikChange", we'd see a simulateneous presence of both parent
and mutated genotype. 

I always discounted it on "cryptic double clone" or simple screw ups.
The frequency was never something to really pay attention to. 

Until this last time. I've screened clones by colony PCR, picked 
positives, sequenced two clones. In BOTH clones, it is definitely 
a mix. A simple mix up can be confidently excluded in this case 
(re-tested, double checked going back to the original colony PCR 
templates, etc, etc). 

Re-transform isolated pure clones without a problem but it's got me
thinking about the mechanism. Why would two different plasmids 
end up in the same cell and persist together all the way through 
a large clone and an overnight culture grown from it? And, how 
to control/limit this occurence? With two clones having the same 
wierd problem, this looks like something systemati

Re: 5x sds sample buffer become brown color while preparing

Dr Engelbert Buxbaum — 2013-04-21T11:58:24

In article <mailman.276.1366318008.10461.methods-zqAmISg3oQ7R7s880joybQ< at >public.gmane.org>, 
sudheer.pbm07-Re5JQEeQqe8AvxtiuMwx3w< at >public.gmane.org says...

Promophenolblue is an indicator with a colour chnage from blue to 
yellow. My guess is that your solution has a pH just around the pKa 
value of bromophenolblue (3.6). Remember that the pH of most buffers 
change upon dilution, because it is the activity, rather than the 
concentation that determines pH. In addition, the high glycerol content 
may also affect the colour. Is SDS actually soluble under these 
conditions?

Re: 5x sds sample buffer become brown color while preparing

Christian Praetorius — 2013-04-20T20:36:55


Two possibilities: Bromophenol blue is virtually insoluble in water.
If the concentration is too high, it stays reddish brown. Try to
dilute it with water to 1x and it should turn blue.
Then it can be a effect of the pH, since bromophenol blue is a pH
indicator as well.

Christian

Re: Any experiences with Geneamp 9700 cycler?

Christian Praetorius — 2013-04-20T20:31:46


We have been using four of the 9700s with these "trays" for years now
without any problems. 

Christian

Re: Any experiences with Geneamp 9700 cycler?

Duncan Clark — 2013-04-19T15:23:14

Historians believe that in newspost <kkq6h5$ilf$1< at >dont-email.me> on Fri, 
19 Apr 2013, DK <dk-ExnEkaPBXRK421ATBNoTQIIY+NxbBFVqXqFh9Ls21Oc< at >public.gmane.org> penned the following 
literary masterpiece:

We use what used to be ABgene tubes, now part of Thermofisher.


It's not going to really matter. Polypropylene melts a long way above 
that so anything approx. will do.


The problem with the thermocouple is that it will be measuring block 
temperature and not the in tube temperature. There will be at least a 
0.5C difference. Just as difficult measuring in the tube because the 
sheer mass of even a tiny thermocouple will pull heat out of the small 
volume in the tube.

A multiplex PCR across all wells and run out on an agarose gel is pretty 
good at testing consistency across a block. Overshoot or undershoot 
isn't usually too much of an issue providing it is consistent across the 
whole block. I don't want a PCR working in the middle of a block and 
failing at all the edges!

Duncan

Re: Any experiences with Geneamp 9700 cycler?

DK — 2013-04-19T01:30:00

Following Mike's advise, I ordered some of the PE's original "trays" 
to go between the block and the tubes. Another opinion, perfectly 
reasonable, is that the kind of tubes one uses really matters. 
What tubes do you use? I used the ones that worked really well
on 2720 model and I don't think that they are made of polypropylene,
for example. PE/ABI claim that plasticware requirements for 9700
and 2700 series are identical. My problem stems from the fact that 
this is most definitely not the case. (Or I am having a bad dream).


Really? I thought 100C is the standard. But I really don't have experience 
with that many cyclers. Good to know it's not a fundamental problem. 


In the initial testing, I stuck a thermocouple wire betwen a block and a 
thin-walled tube jammed in place. *Looked* accurate enough and 
of course I am not about to buy $2000 calibration kit (which just might 
to be the same thing...)  

DK

5x sds sample buffer become brown color while preparing

Sudheer Sangeetham — 2013-04-18T12:06:26

Hello Everyone

I was trying to prepare 5x sds sample buffer and it has become brown color
instead of blue color. Could anyone please tell me what is the reason and
please tell me your suggestions.

The 5x sample buffer recipe

250mM tris-hcl ph 6.8(1M stock)------10ml
10% sds-------------------------4gr
50% glycerol(87%stock)------------------23ml
0.02% Bromphenol blue-----0.008gr
final volume-------------------------40ml with DDh20 including 1.4ml of 0.5
M B-mercaptoethanol but I didnt add here.

Thanking you in advance

Re: Any experiences with Geneamp 9700 cycler?

Duncan Clark — 2013-04-18T09:00:42

Historians believe that in newspost 
<mailman.265.1366133494.10461.methods-zqAmISg3oQ7R7s880joybQ< at >public.gmane.org> on Tue, 16 Apr 2013, 
Michael Sullivan <mlsulliv-63mtpxcE9Cs< at >public.gmane.org> penned the following literary 
masterpiece:

Likewise we have two over at least that period.

Only issues we have had are one board failure and a block failure. No 
issues with the heated lid affecting tubes.

104C is pretty standard for a heated lid.

DK how are you determining overshoot, measurement by thermocouple in a 
tube or watching the temp. The displayed temp is block temp and not tube 
temp which, will always lag behind.

Duncan

Re: RNA isolation from Formaldehyde fixed samples

Shahrzad Jalali — 2013-04-17T19:05:10

I had followed exactly the kit direction to isolate RNA from formalin fixed
paraffin embedded tissue. I am not sure how it will work if you use
formalin fixed tissue...you would probably need to give a try.


On Wed, Apr 17, 2013 at 10:54 AM, Nick Theodorakis <
nick.theodorakis-Re5JQEeQqe8AvxtiuMwx3w< at >public.gmane.org> wrote:

Re: RNA isolation from Formaldehyde fixed samples

Pow Joshi — 2013-04-17T18:39:42


On a related note, I read the Plosone paper that Nick sent, the proteinase
K and heating to 65-70 deg C is the method to digest the bound proteins and
break the methylene crosslinking bridges. I believe it is done in that
order, protease then heat. I was wondering if one could potentially do it
in the reverse, heat first then add a smaller amount of protease? and would
that lead to a better yield ?.... has anyone tried any such combinations,
and theoretically if there could be problems such as RNA hydrolysis or any
others....

I'd appreciate anything you may have to add to this discussion!

Thank you,
Pow

Re: RNA isolation from Formaldehyde fixed samples

Pow Joshi — 2013-04-17T18:03:34



Thank you, both, Engelbert, and  Nick. I will keep these in mind, and look
at the Plosone paper as well. It woudl be awesome if the samples can be
used for real time pcr assays ...
Pow