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Re: Purifying 5' phosphorylated primers for mutagenesis

2009-11-27T06:22:45Z

I'm actually aiming to do a slight site-directed mutagenesis PCR reaction
shortly, I imagined it was as simple as doing a PCR with lower annealing
temperatures and a regular primer with a point mutation.. Am I mistaken?

What protocols do others on this list use for PCR-based mutagenesis? I'm
using KOD hotstart enzyme, which has exonuclease activity. It occurs to me
that it might inhibit any mismatches I might need for mutagenesis to occur,
should I be adding a solvent (such as DMSO) to inhibit exonuclease activity?

Thanks!
Cathal Garvey
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Re: Re: lyophilizing leaf tissue

2009-11-26T23:15:04Z

While I totaly agree that extracting and sending the extract might be easier,
"Total" lipid extraction could be trickier then one would think. You might
watnt to take a look at this link
<http://www.lipidlibrary.co.uk/topics/extract/index.htm> and at the original
Folck papaer <http://www.jbc.org/content/226/1/497.full.pdf+html>.
Yoram

Quoting methods-request@...:

>
> Message: 3
> Date: Thu, 26 Nov 2009 12:34:24 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum@...>
> Subject: Re: lyophilizing leaf tissue
> To: methods@...
> Message-ID: <op.u30gbmaz66vu6s@...>
> Content-Type: text/plain; format=flowed; delsp=yes;
> charset=iso-8859-15
>
> Am 24.11.2009, 09:46 Uhr, schrieb Jeremy Coate <coatej@...>:
>
> > Hi savy methodologists,
> >
> > I need to lyophilize leaf tissue prior to shipping overseas. The tissue
> > will
> > be used for analysis of lipids, which are vulnerable to endogenous
> > lipases,
> > so I am planning to collect the tissue into liquid N2, freeze dry and
> > ship
> > on dry ice to try to ensure that these lipases don't have a chance to do
> > their thing. I have never used a lyophilizer and have concerns that I'm
> > using it incorrectly, and that the samples are thawing.
> >
> > I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on
> > top
>
> You should have somebody show you how to use the instrument correctly.
> That only takes 2 min, rocket science it ain't. Lyophilizers consist of a
> vacuum pump and a cold trap. You connect your deep frozen sample to it,
> and the solvent (water in your case) sublimates. The vapor is condensed in
> the cold trap, which has to be cleaned from time to time. All the heat
> coming into your sample from the environment is used to increase the
> sublimation rate, the heat of evaporation ensures that the sample stays
> frozen until all solvent has been removed.
>
> In your case however, it perhaps would be more appropriate to extract the
> lipids and ship those. See
> @article{Rad-81,
> AUTHOR= {M.S. Radin},
> TITLE= {Extraction of tissue lipids with a solvent of low
> toxicity},
> YEAR= {1981},
> JOURNAL= {Meth. Enymol.},
> PAGES= {5-7},
> VOLUME= {72},
> LANGUAGE= {engl}
> }
> which uses cyclohexane rather than chloroform as in
> @article{Bli-59,
> AUTHOR= {E.G. Bligh and W.J. Dyer},
> TITLE= {A rapid method for total lipid extraction and
> purification},
> YEAR= {1959},
> JOURNAL= {Canadian J. Biochem. Physiol.},
> PAGES= {911-917},
> VOLUME= {37},
> NUMBER= {8},
> LANGUAGE= {engl}
> }
> The trick with all these procedure is to work under nitrogen, to prevent
> lipid peroxidation.
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods@...
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 54, Issue 14
> ***************************************
>

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Re: Purifying 5' phosphorylated primers for mutagenesis

2009-11-26T22:28:16Z

For most cases, just desalted primers work most of the time. Using
purified primers can help if you use very long primers (50+ nt) or are
doing long insertions/deletions. For problematic primers, I like to
buy from Bioneer, which offers reverse phase purification for free.

ho

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Re: lyophilizing leaf tissue

2009-11-26T08:34:24Z

Am 24.11.2009, 09:46 Uhr, schrieb Jeremy Coate <coatej@...>:

> Hi savy methodologists,
>
> I need to lyophilize leaf tissue prior to shipping overseas. The tissue
> will
> be used for analysis of lipids, which are vulnerable to endogenous
> lipases,
> so I am planning to collect the tissue into liquid N2, freeze dry and
> ship
> on dry ice to try to ensure that these lipases don't have a chance to do
> their thing. I have never used a lyophilizer and have concerns that I'm
> using it incorrectly, and that the samples are thawing.
>
> I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on
> top

You should have somebody show you how to use the instrument correctly.
That only takes 2 min, rocket science it ain't. Lyophilizers consist of a
vacuum pump and a cold trap. You connect your deep frozen sample to it,
and the solvent (water in your case) sublimates. The vapor is condensed in
the cold trap, which has to be cleaned from time to time. All the heat
coming into your sample from the environment is used to increase the
sublimation rate, the heat of evaporation ensures that the sample stays
frozen until all solvent has been removed.

In your case however, it perhaps would be more appropriate to extract the
lipids and ship those. See
@article{Rad-81,
AUTHOR= {M.S. Radin},
TITLE= {Extraction of tissue lipids with a solvent of low
toxicity},
YEAR= {1981},
JOURNAL= {Meth. Enymol.},
PAGES= {5-7},
VOLUME= {72},
LANGUAGE= {engl}
}
which uses cyclohexane rather than chloroform as in
@article{Bli-59,
AUTHOR= {E.G. Bligh and W.J. Dyer},
TITLE= {A rapid method for total lipid extraction and
purification},
YEAR= {1959},
JOURNAL= {Canadian J. Biochem. Physiol.},
PAGES= {911-917},
VOLUME= {37},
NUMBER= {8},
LANGUAGE= {engl}
}
The trick with all these procedure is to work under nitrogen, to prevent
lipid peroxidation.
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Re: AKTA FPLC Mixer 925 Problem and Solution

2009-11-26T08:19:02Z

Am 17.11.2009, 08:00 Uhr, schrieb Chalmers <chalmers@...>:

> The mixer is basically a magnetic stirrer. I opened the unit and found
> a little 12 V DC motor that turns the magnet. I was confused at first
> because it worked when I attached a 9V battery. The final explanation
> was that the motor had difficultly getting started, particularly at 4 C,
> but not so bad at room temperature. It worked intermittently for a
> while then failed. It had only 2 ohms resistance between the terminals
> (I suspect it should be 12 ohms).
>
> The ID numbers on the motor specify a custom motor supplied to the AKTA
> manufacturers, so you can not buy one. However, it looked suspiciously
> like a Premotec 990412018105. You can buy one for £47 from RS
> components (the name on the motor is different but the manufacturers
> product code is identical).
>
> The Premotec motor is rated to run at 3600 rpm at 12 V. However, it
> receives only 1.89 V from the Mixer 925 unit. It therefore runs close
> to the specified 600 rpm when drawing power from the Mixer 925.

This is not unusual: the motor is operated with a lower than specified
voltage to reduce the rpm. This is actually a design flaw, for
unfortunately this also reduces torque. Thus the motor may not start,
especially under condition that increase the torque required, like low
temperatures (metal shrinkage in bearing, higher viscosity of lubricants).
The solution is not to replace the motor (which is perfectly fine), but to
build a puls width modulation power supply for it. PWM means that the
motor is operated with impulses instead of DC, which have nominal voltage
(giving full torque). The width of these impulses determines rpm. Any
competent workshop can do that in a day or so, it's actually quite simple.
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Most cited research reagents and kits

2009-11-25T08:48:55Z

An interactive graph showing most cited research reagents and kits that are commercially available. The data is based on recent published journal articles, patents and patent applications. The current research reagent/kits listings are Transfection reagents, RT-PCR, Real-time PCR, RNA purification, cAMP assays, Cytotoxicity assays and Western blot reagents. Please visit: http://www.sciclips.com/sciclips/most-cited-reagents.do?cat=Transfection Reagents&catId=1501
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Re: lyophilizing leaf tissue

2009-11-24T19:13:07Z

Hi Jeremy,

Lyophilizers suppose to fully dry up water content of the sample, so
"thawing" shouldn't be the concern.

lautys

On Tue, Nov 24, 2009 at 9:46 PM, Jeremy Coate <coatej@...> wrote:

> Hi savy methodologists,
>
> I need to lyophilize leaf tissue prior to shipping overseas. The tissue
> will
> be used for analysis of lipids, which are vulnerable to endogenous lipases,
> so I am planning to collect the tissue into liquid N2, freeze dry and ship
> on dry ice to try to ensure that these lipases don't have a chance to do
> their thing. I have never used a lyophilizer and have concerns that I'm
> using it incorrectly, and that the samples are thawing.
>
> I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on
> top_______________________________________________
> Methods mailing list
> Methods@...
> http://www.bio.net/biomail/listinfo/methods
>

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Purifying 5' phosphorylated primers for mutagenesis

2009-11-24T14:12:50Z

I'm doing some site-directed mutagenesis with a kit from NEB. It is
recommended in the kit directions that the 5' phosphorylated primers used
for the reaction are purified either using RP-HPLC or PAGE.

My issue is that these purification methods are *really* expensive. (I've
been getting my primers through Invitrogen, but I've also looked at a few
other places and it's the same story.) Has anyone had success using primers
purified another (less expensive) way or done the purification in house?

Thank you,

Haley, University of Washington
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Re: Looking for Hot Bonnet for PTC150

2009-11-24T08:20:52Z

Historians believe that in newspost
<mailman.351.1259021735.1133.methods@...> on Mon, 23 Nov 2009,
Matthew Skeels <mskeels@...> penned the following literary
masterpiece:
>I was wondering if someone could help me find a hot bonnet for a MJR
>PTC150? [used or new?]

You maybe surprised but Ebay is worth a look!

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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Re: Looking for Hot Bonnet for PTC150

2009-11-24T06:16:30Z

Matthew Skeels <mskeels@...> writes:

> I was wondering if someone could help me find a hot bonnet for a MJR
> PTC150? [used or new?]

Bio-rad now maintains MJ Research instruments; I believe they will
have this part.

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lyophilizing leaf tissue

2009-11-24T05:46:40Z

Hi savy methodologists,

I need to lyophilize leaf tissue prior to shipping overseas. The tissue will
be used for analysis of lipids, which are vulnerable to endogenous lipases,
so I am planning to collect the tissue into liquid N2, freeze dry and ship
on dry ice to try to ensure that these lipases don't have a chance to do
their thing. I have never used a lyophilizer and have concerns that I'm
using it incorrectly, and that the samples are thawing.

I'm using a Labconco FreeZone 18 freeze dryer with a bulk dryer unit on top_______________________________________________
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qPCR 2010 Event - Call for Abstracts

2009-11-24T04:47:10Z

qPCR 2010 Event - Call for Abstracts

www.qPCR2010-Vienna.net

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

BioEPS GmbH is organizing the qPCR 2010 Event taking place April 7th –
9th, 2010 in Vienna, Austria. Scientists from all around the world
will come to exchange ideas, share experiences, and discuss the
exciting future of the perhaps most powerful analytical technology
ever developed in the life sciences area – the quantitative real-time
polymerase chain reaction (qPCR).

We have the pleasure to announce the Call for Abstracts for the qPCR
2010 Event !

Finally we want to present 40 scientific talks from international
scientists and diagnostic companies in the qPCR field who will show
their latest research findings and newest technologies. The focus of
the qPCR 2010 Event will be “The ongoing evolution of qPCR”
representing all new and emerging techniques, applications and data
analysis methods.

Deadline is 31st January 2010 => http://submission.qPCR2010-Vienna.net

Talk topics:
--------------

MIQE and QM strategies in qPCR
The MIQE guidelines: minimum information for publication of
quantitative real-time PCR experiments. Following these guidelines
will encourage better experimental practice, allowing more reliable
and unequivocal interpretation of qPCR results. QM strategies in real-
time PCR to guarantee better and more valid results.
Keynote speaker: Prof. Stephen Bustin

High throughput quantitative PCR – digital PCR
384 well applications, new high throughput platforms, droplet PCR,
qPCR robotics, digital PCR, gene expression real-time RT-PCR arrays
(mRNA and microRNA), quantitative multiplexing, …
Keynote speaker: Prof. Mikael Kubista, Dr. Philip Day, Dr. Ken
Livak

HRM – High Resolution Melting
SNP analysis, HRM = high resolution melt applications, Epigenetics,
methylation markers, HRM platform comparison, etc …
Keynote Speaker Prof. Carl Wittwer, Prof. Claudio Orlando

Circulating nucleic acids
Analysis of circulating RNAs and DNA and microRNAs as diagnostic and
prognostic marker, …
Speaker: Dr. Pamela Pinzani, Dr. Alfred Schöller, Dr. Jim Huggett

Single–cell qPCR
single-cell sampling, pre-amplification techniques, laser micro-
dissection, sub-cellular PCR, micro-manipulation of cell clusters,
cellular micro injection, FACS spotting, single cell handling, pre-
amplification, …
Keynote speaker: Dr. Michael W. Pfaffl, Dr. Anders Stahlberg

RNAi – microRNA – siRNA Applications – miRNA normalisation
RNAi mechanism, microRNA extraction, qRT-PCR technologies to detect
microRNA, microRNA normalisation strategies, siRNA applications in
combination with qRT-PCR, microRNA targets and microRNA precursors,
new siRNA manipulation and microRNA technologies,
Keynote speaker: Prof. Jo Vandesompele, Dr. Mirco Castoldi

qPCR BioStatistics & BioInformatics
software applications, data mining, calculation of relative
expression, primer and probe design on mRNA and microRNA level, real-
time PCR efficiency determination, mathematical modelling,
multivariate expression profiling, statistics in real-time PCR, data
management, multiway expression profiling, multiple regression
analysis, 3D data visualization, ...
Speaker: Dr. Ales Tichopad, Dr. Jan Hellemans, Dr. Anders
Bergkvist

An online registration and abstract submission software CONFTOOL is
available here => http://registration.qPCR2010-Vienna.net

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

About qPCR:
Using qPCR the amount of target nucleic acid in a complex sample can
be determined with high precision, great accuracy, excellent
specificity and the ultimate sensitivity of detecting a single
molecule. The technique has revolutionized all molecular sciences and
diagnostic applications. Conference presentations will include MIQE
guidelines & QM strategies in qPCR, high performance nucleic acid
extraction, single-cell applications, Epigenetics & High-Resolution-
Melt analysis, circulating nucleic acids, and application involving
RNAi and microRNA.
Further developments of qPCR technology will be presented include
improved instrumentation, miniaturization, high throughput platforms,
cost efficacy, validity, flexibility, quality assessment and reliable
Cq calculations, expression data comparisons, and interpretation.
Today there is no field in the life sciences research, molecular
biology and diagnostics areas that has not introduced qPCR technology
for nucleic acid analysis. The combination with reverse transcription
enables determination of mRNA, microRNA and widely opens the window
for “Transcriptomics” – the first step of quantitative “Gene
Expression Profiling” and “Functional Genomics”.
An Industrial Exhibition will take place parallel to the symposium,
with 32 leading biotechnology companies presenting their latest
developments, including real-time PCR cyclers, NA extraction robots,
consumables, new fluorescence dyes, NA detection and amplification
chemistries, as well as real-time PCR data analysis software.

For more information about the qPCR 2010 event contact Dr. Martina
Reiter Martina.Reiter@...

Hope to meet you in April in Vienna!

Michael Pfaffl
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Looking for Hot Bonnet for PTC150

2009-11-23T15:32:11Z

Hello,

I was wondering if someone could help me find a hot bonnet for a MJR
PTC150? [used or new?]

Cheers,
MS
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Re: Sequential restriction digest

2009-11-22T10:53:25Z

Dear all!
Of course, using isoschizomers or hifi enzymes are an interesting idea,
but are either time consuming or expensive for only one application. I
have therefore tried the low-salt-buffer-first suggestion, and it really
worked great! Complete digest, no waste of agarose and extraction kits,
simply two-step-single-tube ;-)
Thank you all very much for your suggestions!
Niko

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Fwd: RNA ligation

2009-11-21T16:42:34Z

You're right Nick, it's chemically-decapped mRNA. You knock the 5'
phosphate off with tobacco acid pyrophosphatase - it's standard to most 5'
'RACE protocols. On the question of whether its technically correct to
refer to a decapped mRNA moiety as 'mRNA', I don't see any harm since an
uncapped mRNA can still function as a messenger RNA in that it can generate
protein products (albeit reduced in the case of cap-dependent translation
but not in the case of cap-independent translation).

---------- Forwarded message --------
From: Nick Theodorakis <nick.theodorakis@...>
Date: 2009/11/21
Subject: Re: RNA ligation
To: methods@...

On Nov 20, 9:58 pm, Aawara Chowdhury <aaw...@...>
wrote:

> In <4e7e0a8e-85b3-4eda-9190-7740ce3fc...@...>,
> Nick Theodorakis <nick.theodora...@...> wrote:
>
>
>
> >> Won't work for mRNA - there is no free 5'phosphate.
>
> > He specified that his RNA population is uncapped and phosphorylated in
> > his original post. I don't know how it got that way, but I can imagine
> > at least one scenario.
>
> Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs
> to be capped.
>

Well, whatever. I'll just assume that the OP knows something about the
RNA he has. But consider it could be:

1) prokaryotic or viral origin
2) mRNA that was chemically decapped or partially degraded
3) he could be looking for degradation intermediates.

If you want to get nitpicky I suppose you can call 2) or 3) "mRNA-
derived RNA fragments" or something, if that will make you happy.

Nick

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Re: RNA ligation

2009-11-20T19:35:57Z

On Nov 20, 9:58 pm, Aawara Chowdhury <aaw...@...>
wrote:

Re: RNA ligation

2009-11-20T18:58:13Z

In <4e7e0a8e-85b3-4eda-9190-7740ce3fc37e@...>,
Nick Theodorakis <nick.theodorakis@...> wrote:

>>
>> Won't work for mRNA - there is no free 5'phosphate.
>>
>
> He specified that his RNA population is uncapped and phosphorylated in
> his original post. I don't know how it got that way, but I can imagine
> at least one scenario.

Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs
to be capped.

AC
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Re: RNA ligation

2009-11-20T04:47:29Z

On Nov 20, 6:19 am, Aawara Chowdhury <aaw...@...>
wrote:

> In <63e4a4c6-9734-4886-baba-dab9a41e7...@...>,
> Nick Theodorakis <nick.theodora...@...> wrote:
>
> >> Yes, they'll both be single stranded. ?One will be the mRNA that I want to
> >> tag (at the 5' end), the other will be a ss oligo. ?My concern is that the
> >> oligo may ligate to the 3' end of the mRNA but I guess this will depend on
> >> how I have the oligo modified? ?Is it sensible to assume that if it (the
> >> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
> >> the exposed 5' phosphate on the mRNA and NOT the 3' OH??
>
> > That sounds sensible. If the oligo has no terminal phosphates and the
> > RNA is 5' phosphorylated, then the only reactions that should happen
> > are ligation of the oligo to the 5'end of the RNA .. [deleted]..
>
> Won't work for mRNA - there is no free 5'phosphate.
>

He specified that his RNA population is uncapped and phosphorylated in
his original post. I don't know how it got that way, but I can imagine
at least one scenario.

Nick

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Re: RNA ligation

2009-11-20T03:19:26Z

In <63e4a4c6-9734-4886-baba-dab9a41e76b7@...>,
Nick Theodorakis <nick.theodorakis@...> wrote:

>> Yes, they'll both be single stranded. ?One will be the mRNA that I want to
>> tag (at the 5' end), the other will be a ss oligo. ?My concern is that the
>> oligo may ligate to the 3' end of the mRNA but I guess this will depend on
>> how I have the oligo modified? ?Is it sensible to assume that if it (the
>> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
>> the exposed 5' phosphate on the mRNA and NOT the 3' OH??
>
> That sounds sensible. If the oligo has no terminal phosphates and the
> RNA is 5' phosphorylated, then the only reactions that should happen
> are ligation of the oligo to the 5'end of the RNA .. [deleted]..

Won't work for mRNA - there is no free 5'phosphate.

AC
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Re: RNA ligation

2009-11-20T03:11:11Z

In <mailman.322.1258653708.1133.methods@...>,
Chris McDermott-Roe <cjmcdermottroe@...> wrote:

> Hi,
>
> Yes, they'll both be single stranded. One will be the mRNA that I want to
> tag (at the 5' end), the other will be a ss oligo. My concern is that the
> oligo may ligate to the 3' end of the mRNA but I guess this will depend on
> how I have the oligo modified? Is it sensible to assume that if it (the
> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
> the exposed 5' phosphate on the mRNA and NOT the 3' OH??

That will NOT work. mRNAs do not have a free 5' phosphate. As you may
recall, mRNAs are capped with a 7-methylguanosine cap which is covalently
linked to the first transcribed nucleotide by a 5'-5' triphosphate bond.

There are mammalian/eukaryotic and viral decapping enzymes, but I don't
know of any that work on all mRNAs in vitro, or in vivo.

AC
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Re: RNA ligation

2009-11-19T20:23:41Z

On Nov 19, 5:06 am, Chris McDermott-Roe
<cjmcdermott...@...> wrote:

> Hi,
>
> Yes, they'll both be single stranded. One will be the mRNA that I want to
> tag (at the 5' end), the other will be a ss oligo. My concern is that the
> oligo may ligate to the 3' end of the mRNA but I guess this will depend on
> how I have the oligo modified? Is it sensible to assume that if it (the
> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
> the exposed 5' phosphate on the mRNA and NOT the 3' OH??
>
>

That sounds sensible. If the oligo has no terminal phosphates and the
RNA is 5' phosphorylated, then the only reactions that should happen
are ligation of the oligo to the 5'end of the RNA or circularization
on the RNA. It seems to me you might be able to prevent the latter by
pCp tailing the RNA first if you think that will be a problem.
Disclaimer: I haven't tried any of these reactions myself. There are a
couple of references mentioned in the NEB I link I posted earlier that
might possibly be of use.

Nick

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How long for DNA to reach nucleus?

2009-11-19T17:30:36Z

Did you ever get an answer for this question?
--
Ketna Volcy, Ph.D.
Post-doctoral Fellow
University of Pennsylvania Medical Center
319 Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104
215-898-3846

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Re: Sequential restriction digest

2009-11-19T15:38:52Z

I'm being forced to do likewise, with XhoI and SacI. It's a lot of
frustration and sequential purification (my method of choice, as Gel
Extractions are even less reliable) is my currently favoured method.

However, a quick look at the Fermentas FastDigest range of enzymes shows
that their speed (~15mins) is the least of their virtues; they all cut in
the same buffer, which incidentally contains loading dye for gel loading as
well.

They're expensive compared to the regular range, but I consider the extra
cost well worth it for the time saved and the satisfaction of a job done
quickly.

2009/11/19 Nikola Wenta <Nikola.Wenta@...>

> Dear all,
> I want to do a SacI/SalI digest of my plasmid in order to subclone my
> insert (1.2 kb) into another vector. According to NEB, the REs need
> different buffers, so I would have to do a sequential digest. Does
> anyone have a working protocol for sequential digests? Particularly I
> would like to know how to proceed once the first digest i.e. with SacI
> has linearized my original plasmid. Would you run a preparative agarose
> gel and cut the band out, or would you precipitate the DNA and resuspend
> the resulting DNA pellet in the buffer for the subsequent SalI digest?
> Thank you for any suggestions!
> Niko
>
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> may still contain software viruses, which could damage your computer
> system:
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> University of Nottingham may be monitored as permitted by UK legislation.
>
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Re: Sequential restriction digest

2009-11-19T14:35:21Z

For the particular combination you are talking about, both the pH and
the salt concentrations of the buffers differ.

What I would probably do would be to digest first with Sac I in NEB
buffer 1. When you are satisfied the digest is complete (for example
by running an aliquot on a gel), I would then add 1/10 volume (that
is the final volume you expect with the added buffer and enzyme) NEB
buffer 3 and Sal I. For this combination of enzymes, this strategy
should work because both the buffer concentration and salt
concentration of NEB1 are very low and will have little impact on the
reaction conditions following addition of the NEB buffer 3.

If you do decide to purify following the first digest, don't waste
time prepping from a gel. In my mind, the only reason to gel purify
is if you only want one fragment from a digest that gives multiple
fragments. Precipitation is fine (and recovery is usually pretty good
unless you are starting with very small amounts of DNA). You can also
use a clean up type product (there are many)-- these are quick and
convenient.

Hope this helps.

Mike

On Nov 19, 2009, at 2:46 PM, Nikola Wenta wrote:

> Dear all,
> I want to do a SacI/SalI digest of my plasmid in order to subclone my
> insert (1.2 kb) into another vector. According to NEB, the REs need
> different buffers, so I would have to do a sequential digest. Does
> anyone have a working protocol for sequential digests? Particularly I
> would like to know how to proceed once the first digest i.e. with SacI
> has linearized my original plasmid. Would you run a preparative
> agarose
> gel and cut the band out, or would you precipitate the DNA and
> resuspend
> the resulting DNA pellet in the buffer for the subsequent SalI digest?
> Thank you for any suggestions!
> Niko
>
> This message has been checked for viruses but the contents of an
> attachment
> may still contain software viruses, which could damage your
> computer system:
> you are advised to perform your own checks. Email communications
> with the
> University of Nottingham may be monitored as permitted by UK
> legislation.
>
> This message has been checked for viruses but the contents of an
> attachment
> may still contain software viruses which could damage your computer
> system:
> you are advised to perform your own checks. Email communications
> with the
> University of Nottingham may be monitored as permitted by UK
> legislation.
>
> _______________________________________________
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Re: Sequential restriction digest

2009-11-19T13:36:59Z

I generally try to avoid purification of the DNA between digests, as you
lose a lot of it in the process. The way I've done sequential digest is to
cut with the enzyme which uses a low salt buffer, and then spike in salt to
adjust to the concentration of the second enzyme's buffer. Sometimes you
have to fudge a bit and use a non-optimal buffer for the first one.

An even simpler way is to use an isoschizomer for one of the enzymes so they
cut in the same buffer. In the past, I've had some finicky sequential
digests that worked beautifully if I just switched enzymes. I would highly
recommend doing this.

Adam

On Thu, Nov 19, 2009 at 2:46 PM, Nikola Wenta <Nikola.Wenta@...
> wrote:

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Sequential restriction digest

2009-11-19T12:46:33Z

Dear all,
I want to do a SacI/SalI digest of my plasmid in order to subclone my
insert (1.2 kb) into another vector. According to NEB, the REs need
different buffers, so I would have to do a sequential digest. Does
anyone have a working protocol for sequential digests? Particularly I
would like to know how to proceed once the first digest i.e. with SacI
has linearized my original plasmid. Would you run a preparative agarose
gel and cut the band out, or would you precipitate the DNA and resuspend
the resulting DNA pellet in the buffer for the subsequent SalI digest?
Thank you for any suggestions!
Niko

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University of Nottingham may be monitored as permitted by UK legislation.

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Re: RNA ligation

2009-11-19T02:06:14Z

Hi,

Yes, they'll both be single stranded. One will be the mRNA that I want to
tag (at the 5' end), the other will be a ss oligo. My concern is that the
oligo may ligate to the 3' end of the mRNA but I guess this will depend on
how I have the oligo modified? Is it sensible to assume that if it (the
oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
the exposed 5' phosphate on the mRNA and NOT the 3' OH??

hope this makes sense

Chris

2009/11/19 Nick Theodorakis <nick.theodorakis@...>

> On Nov 18, 11:35 am, Chris McDermott-Roe
> <cjmcdermott...@...> wrote:
> > Hi everyone,
> >
> > I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped
> but
> > not dephosphorylated) of an mRNA population. Can anybody tell me the
> best
> > way of doing this?
> >
> > many thanks
> >
> > Chris
>
> Are they both single stranded? I think T4 RNA ligase will do that
> reaction.
>
> See:
> <http://www.neb.com/nebecomm/products/productM0204.asp>
>
> Nick
>
> --
> Nick Theodorakis
> nick_theodorakis@...
> contact form:
> http://theodorakis.net/contact.html
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Re: RNA ligation

2009-11-18T17:54:25Z

On Nov 18, 11:35 am, Chris McDermott-Roe
<cjmcdermott...@...> wrote:
> Hi everyone,
>
> I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped but
> not dephosphorylated) of an mRNA population. Can anybody tell me the best
> way of doing this?
>
> many thanks
>
> Chris

Are they both single stranded? I think T4 RNA ligase will do that
reaction.

See:
<http://www.neb.com/nebecomm/products/productM0204.asp>

Nick

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RNA ligation

2009-11-18T08:35:18Z

Hi everyone,

I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped but
not dephosphorylated) of an mRNA population. Can anybody tell me the best
way of doing this?

many thanks

Chris
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Re: AKTA FPLC Mixer 925 Problem and Solution

2009-11-18T02:59:37Z

Chalmers <chalmers@...> wrote in
news:mailman.308.1258476303.1133.methods@...:

> This is not exactly a method but could save you money.
>
> We have an AKTA FPLC with the Mixer 925 unit. We don't have a service
> contract so when the mixer unit stopped working and the system
> provided a short-circuit warning message, we asked the price of a new
> one. We were quoted £10,000, which is half the price of the whole
> system. Presumably this is a joke, designed to force you to call in
> the engineer and then get a service contract, perhaps.
>
> The mixer is basically a magnetic stirrer. I opened the unit and
> found a little 12 V DC motor that turns the magnet. I was confused at
> first because it worked when I attached a 9V battery. The final
> explanation was that the motor had difficultly getting started,
> particularly at 4 C, but not so bad at room temperature. It worked
> intermittently for a while then failed. It had only 2 ohms resistance
> between the terminals (I suspect it should be 12 ohms).
>
> The ID numbers on the motor specify a custom motor supplied to the
> AKTA manufacturers, so you can not buy one. However, it looked
> suspiciously like a Premotec 990412018105. You can buy one for £47
> from RS components (the name on the motor is different but the
> manufacturers product code is identical).
>
> The Premotec motor is rated to run at 3600 rpm at 12 V. However, it
> receives only 1.89 V from the Mixer 925 unit. It therefore runs close
> to the specified 600 rpm when drawing power from the Mixer 925.
>
> It takes about 5 minutes to change the motor using simple tools, and
> we saved a significant amount of money.
> This message has been checked for viruses but the contents of an
> attachment may still contain software viruses, which could damage your
> computer system: you are advised to perform your own checks. Email
> communications with the University of Nottingham may be monitored as
> permitted by UK legislation.
>
> This message has been checked for viruses but the contents of an
> attachment may still contain software viruses which could damage your
> computer system: you are advised to perform your own checks. Email
> communications with the University of Nottingham may be monitored as
> permitted by UK legislation.
>

Thanks. This is from home, and I don't know by heart whether we have
that system, or another. Neither do I know whether that motor would be
available in the US.

I do know that I could replace the power supply on the Compaq computer
that runs our HPLC after it quit. About US$38 on Ebay.

--
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Han
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AKTA FPLC Mixer 925 Problem and Solution

2009-11-17T04:00:21Z

This is not exactly a method but could save you money.

We have an AKTA FPLC with the Mixer 925 unit. We don't have a service
contract so when the mixer unit stopped working and the system
provided a short-circuit warning message, we asked the price of a new
one. We were quoted £10,000, which is half the price of the whole
system. Presumably this is a joke, designed to force you to call in
the engineer and then get a service contract, perhaps.

The mixer is basically a magnetic stirrer. I opened the unit and
found a little 12 V DC motor that turns the magnet. I was confused at
first because it worked when I attached a 9V battery. The final
explanation was that the motor had difficultly getting started,
particularly at 4 C, but not so bad at room temperature. It worked
intermittently for a while then failed. It had only 2 ohms resistance
between the terminals (I suspect it should be 12 ohms).

The ID numbers on the motor specify a custom motor supplied to the
AKTA manufacturers, so you can not buy one. However, it looked
suspiciously like a Premotec 990412018105. You can buy one for £47
from RS components (the name on the motor is different but the
manufacturers product code is identical).

The Premotec motor is rated to run at 3600 rpm at 12 V. However, it
receives only 1.89 V from the Mixer 925 unit. It therefore runs close
to the specified 600 rpm when drawing power from the Mixer 925.

It takes about 5 minutes to change the motor using simple tools, and
we saved a significant amount of money.
This message has been checked for viruses but the contents of an attachment
may still contain software viruses, which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.

This message has been checked for viruses but the contents of an attachment
may still contain software viruses which could damage your computer system:
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a queston

2009-11-14T10:39:58Z

Dear colleagues
I appreciate if anybody can help me in the following problem:
I used Lpp'-ompA system (developed by Georgio et al., 1996) for surface display of a bacterial metallothioneine fused to chitin binding domain and 6Xhis-tag (on the surface of E.coli BL21). The length of this passenger protein is approximately 15kDa. We did metal binding assay using whole cell and we measured adsorption using a solution of cadmium Cd(NO3)2 in Tris-Hcl pH7.0. But it didn't show any adsorption. Hydropathy plot showed the passenger proteins are hydrophile and also it didn't show any transmembrane domain. I would like to know why does not this protein show any adsorption?
I appreciate your help.
Best Wishes
Reza

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N-acetyl cysteine treatment

2009-11-09T16:11:26Z

Hi Everyone,

I am trying to establish the protocol for the treatment of mice with
N-acetyl cysteine (NAC) dissolved in drinking water.

Does someone knows how often the NAC water has to be replaced with the
fresh one (i.e how stable is NAC aqueous solution) and whether the
acidic pH of the solution should be adjusted?

Thank you in advance.

Beata
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Re: Mold problem in 4°C room

2009-11-06T03:01:07Z

Dear Thomas,

The best, ideal and perfect way to abolish this problem is to fumigate your
4 degree/cold room. There are different ways to fumigate the best way is
KMnO4 (Potassium permanganate) + Formaldehyde (the amount of addition will
depend on size of your room) just i am illustrating the protocol

1. Vacate your room completely (the penetration and disinfection power is so
high it can kill even rigid spores)

2. for room of 10X10 feet you can use minimum of 500g of KMnO4 + 500ml of
Formaldehyde (when both are away they wont react, as soon as you mix it
fumes as volcano you should no be in the room and the surroundings, you
should run away from the room)

3. Seal the room to avoid the fumes to come out of the room

4. Wait for min 12 hours to act completely and efficiently

5. Wet the cloth with ammonia of 500ml and spread in the room for minimum of
6-7 hours to neutralize the fumes

6. Wipe the things with some disinfectant to clean and you can start your
work, I promise you won’t see any single mold for at least next 6 months and
year if you keep clean.

The only disadvantage is when fumigation is going on you can’t sit or work
for one day, maybe you can do this on Saturday night, So that it will be
ready for Monday. This is the usual method we do in CLASS-1 rooms and
CLASS-II, don’t look at the fumes or inhale and don’t near or it will harm
you lot take guidance from your professor and seniors while doing this. Any
thing else please feel free to contact.

--
Yours Sincerely,
*Naveen Vankadari*
Lab No: N209
Graduate Student,
Institute of Molecular & Cell Biology,
ACADEMIA SINICA,
128 Academia Road,section-2,
Nankang, Taipei-115
TAIWAN
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RE: Mold problem in 4°C room

2009-11-05T15:55:17Z

Mold grows in cold rooms because moisture collects on surfaces before being picked up by the evaporator coil on the refrigeration system. Many newer cold-rooms have a drying system on them to prevent the build-up of mold. They are expensive.

In addition people who store live culture in the walk-in coolers have the most problem, because many volatile compounds can be effectively utilized by mold, and particular on wooden and paper surfaces. It is best to keep living culture wrapped or sealed tightly and to keep organic volatiles such as acetic acid, ammonia, amines, sulfides, etc sealed tightly.

One can deal with mold as described below. If the wooden surfaces in the room are wood they have been varnished, the mold and moisture eventually destroys this and a surface such as Stainless steel or polyurethane treated wood may be preferable. Corion or stainless steel works well for table tops.

One very common cause of mold is an improperly working fan-coil unit (the aspect of the machine that transfers air across the evaporator coil). This unit is supposed to pick up moisture and deliver that moisture to its condensate pan, were the condensate is rapidly drained. The condensate pan must be level on the outside edges and drain to a center position and quickly out of the cold-room. It is a frequent occurrence that the fan-coil unit is not doing this properly, the condensate tray builds up water and eventually waves form of the surface. The tops of these waves are picked up by lateral airflow and blow out the front of the unit as aspirate, basically causing all the surfaces in the walk-cooler to become permanently wet. Paper is a most effective collector of this moisture.

-Causes of this can be:
--Microbial clogging of the drain line.
--A unit that is out of balance and vibrating excessively (such as a bad fan-motor bearing)
--With volatile acids - the corrosion of the evaporator coil or the drain pan itself.
--Other clogging agents.
--Improperly installed or improperly working cooling fans (erratic icing over as another symptom)
--A drain pan that is not properly leveled to expel excess water or blockage of the drain line for any other reason.

Other causes:
--Door left open excessively
--Door seals are no longer separating outside (moist) air from inside (cold) air.

A properly working cooler should not grow mold quickly, I cleaned out a cooler back in 1996 that had papered materials from 1984 stored on its shelf, most items did not have great amounts of mold on them.

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