Methods Digest, Vol 48, Issue 21-EDTA solution becomes slurry

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Thu May 28 23:11:07 EST 2009


Use equivalent amount of solid NaOH pellets (one by one) instead of solution
while required amount of Tris.Cl in water is being continuousely stirred on
a megnetic stirrer with pH meter electrode dipped in.

On 5/28/09, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. Re: expression of a subunit (DK)
>   2. qPCR NEWS May 2009 - focus on RNA integrity
>      (Editor www.Gene-Quantification.info)
>   3. EDTA solution becomes slurry (Gourvendu Saxena)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 27 May 2009 19:46:43 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: expression of a subunit
> To: methods from net.bio.net
> Message-ID: <gvk594$inq$1 from news.albasani.net>
>
> In article <mailman.295.1243367083.21502.methods from net.bio.net>, Azam
> Rahimpour <rahimpour_a from yahoo.com> wrote:
> >hi
> >I am going to express a mutant subunit of a multimeric complex protein in
> > mammalian cells , this subunit is expected to incorporate in the whole
> protein
> > complex, I dont know if attachment of small tags like his tag or HA tag
> would
> > disrupt its structure and prevent its incorporation in a complex. does
> any one
> > has any experience?
>
> It's protein dependent. Sometimes it does and sometimes it doesn't.
> You'll have to try and find out. Try both N- and C-terminal tags.
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 28 May 2009 03:58:07 -0700 (PDT)
> From: "Editor www.Gene-Quantification.info"
>        <editor from gene-quantification.info>
> Subject: qPCR NEWS May 2009 - focus on RNA integrity
> To: methods from net.bio.net
> Message-ID:
>        <a06a3042-af9a-4cfe-8187-3320aeb64ffe from o18g2000yqi.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> qPCR NEWS May 2009 - focus on RNA integrity
> ------------------------------------------------------------
>
>
>
> Dear researcher,
> dear Gene Quantification page reader,
>
> Our newsletter informs about the latest news in quantitative real-time
> PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene
> Quantification homepage. The focus of this newsletter issue is:
>
> - The MIQE Guidelines Minimum Information for Publication of
> Quantitative Real-Time PCR Experiments
> - RNA integrity - latest papers
> - PowerNest - illuminating error in qPCR experiment design
> - new TAG CLOUD for better navigation =>
> http://directory.gene-quantification.info/
> - New qPCR workshop modules at the TATAA Biocenter Germany =>
> http://tataa.gene-quantification.info/
> - European wide qPCR application workshops =>  register now !
>
>
>
> --------------------------------------------------------------------------------
>
> The MIQE Guidelines Minimum Information for Publication of
> Quantitative Real-Time PCR Experiments
>
> Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans,
> Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W.
> Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer
> Clinical Chemistry 2009, 55(4): 611-622
>
> => http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf
>
> BACKGROUND:  Currently, a lack of consensus exists on how best to
> perform and interpret quantitative real-time PCR (qPCR) experiments.
> The problem is exacerbated by a lack of sufficient experimental detail
> in many publications, which impedes a reader's ability to evaluate
> critically the quality of the results presented or to repeat the
> experiments.
>
> CONTENT:  The Minimum Information for Publication of Quantitative Real-
> Time PCR Experiments (MIQE) guidelines target the reliability of
> results to help ensure the integrity of the scientific literature,
> promote consistency between laboratories, and increase experimental
> transparency. MIQE is a set of guidelines that describe the minimum
> information necessary for evaluating qPCR experiments. Included is a
> checklist to accompany the initial submission of a manuscript to the
> publisher. By providing all relevant experimental conditions and assay
> characteristics, reviewers can assess the validity of the protocols
> used. Full disclosure of all reagents, sequences, and analysis methods
> is necessary to enable other investigators to reproduce results. MIQE
> details should be published either in abbreviated form or as an online
> supplement.
>
> SUMMARY:  Following these guidelines will encourage better
> experimental practice, allowing more reliable and unequivocal
> interpretation of qPCR results.
>
> =>  http://miqe.gene-quantification.info/
>
>
>
> --------------------------------------------------------------------------------
>
> As part of the MIQE guidelines - chapter 5.1 - the RNA quality control
> is an essential step in the quantification process.
>
> 5. Nucleic acid quality control
> 5.1. RNA samples
> Since it is advisable to use approximately the same amount of RNA for
> cDNA synthesis when comparing different samples, quantification of RNA
> in extracted samples is important. However, there are several
> procedures in common use, including spectrophotometry (Nanodrop),
> microfluidic analysis (Agilent BioAnalyser, BioRad Experion),
> capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye
> detection (Ribogreen); all produce different results making it unwise
> to compare data obtained using the different methods. The preferred
> method for RNA quantity determination uses fluorescent RNA binding
> dyes, for example RiboGreen, which are best for the detection of low
> target concentrations. In any case, it is advisable to measure all
> samples using one method only and to report this
> information.  ... ... ...
>
> latest papers  => http://www.gene-quantification.de/rna-integrity2.html
>
> - Reverse transcription-quantitative polymerase chain reaction:
> description of a RIN-based algorithm for accurate data normalization.
> - Time course analysis of RNA stability in human placenta.
> - Evaluation of isolation methods and RNA integrity for bacterial RNA
> quantitation.
> - A comparison and evaluation of RNA quantification methods using
> viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration
> range.
> - Validation of lab-on-chip capillary electrophoresis systems for
> total RNA quality and quantity control.
> - Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to
> enhance expression analysis from formalin fixed paraffin embedded
> (FFPE) materials.
> - Optimization of the method of RNA isolation from paraffin blocks to
> assess gene expression in breast cancer.
> - Measuring microRNAs: comparisons of microarray and quantitative PCR
> measurements, and of different total RNA prep methods.
> - Focus on RNA isolation: obtaining RNA for microRNA (miRNA)
> expression profiling analyses of neural tissue.
> - Systematic analysis of microRNA expression of RNA extracted from
> fresh frozen and formalin-fixed paraffin-embedded samples.
> - Stability of RNA isolated from post-mortem tissues of Atlantic
> salmon (Salmo salar L.)
> - Prediction of qualitative outcome of oligonucleotide microarray
> hybridization by measurement of RNA integrity using the 2100
> Bioanalyzer capillary electrophoresis system.
> - Removal of contaminating DNA from commercial nucleic acid extraction
> kit reagents.
>
>
>
> --------------------------------------------------------------------------------
>
> PowerNest -  illuminating error in qPCR experiment design
>
> PowerNest is a software tool enabling experimenters to explore the
> effect of sampling on noise propagation throughout qPCR assays.  The
> sampling process is assumed to be comprised of a number of levels; the
> acquisition of a sample and the preparation of extracted material,
> reverse-transcription of the mRNA, and the qPCR itself.  Given a small
> set of data, representative of a larger assay, the error at each stage
> of the experiment is profiled using a nested-ANOVA.
> Armed with this information, PowerNest allows the experimenter to
> explore the effects of modifications to the experimental design on the
> expected total error of the assay.  When given the financial cost of
> replicates at each level, PowerNest will calculate a cost-optimal
> sampling-plan, delivering an experiment design that will minimise
> processing error and maximise the statistical resolution of the assay.
> The software is temporarily undergoing final testing, during which
> time it has been made available as a free download =>
> http://www.gene-quantification.de/main-bioinf.shtml#powernest
>
> PowerNest Poster => http://www.gene-quantification.de/powernest-poster.pdf
>
>
>
> --------------------------------------------------------------------------------
>
> Upcoming Events World-wide academic and commercial qPCR Events
> http://events.gene-quantification.info/
>
> Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars,
> qPCR Education Program, etc.
> Please submit your qPCR event here  =>  events from gene-
> quantification.info
>
>
>
> --------------------------------------------------------------------------------
>
> qPCR WORKSHOP
>
> BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops
>
> At the TATAA Biocenter Germany we offer qPCR application workshops, a
> 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module.  All
> courses are held regularly in Göteborg, Sweden, in English and in
> Freising-Weihenstephan, Germany, in German and English, and in Prague,
> Czech Republic in English and Czech.
> Depending on the occasion the workshop language and the different
> prices may apply. Further customized workshops and specialized
> trainings will be held as well across Europe and world-wide.
> TATAA Biocenter Germany workshops are held in cooperation with BioEPS
> GmbH, located at the campus of the Technical University of Munich, in
> Freising-Weihenstephan, very close to the Munich Airport (MUC). For
> more information and registration, please see our web page:
> => http://TATAA.gene-quantification.info/
>
>
> Course Occasions 2009:
>
> 3-day qPCR Core Module  (Mon. - Wed.)
> 2-day BioStatistics Module (Thu. - Fri.)
> 3-day single-cell qPCR Module  (Mon. - Wed.)
> 3-day microRNA Module   (Mon. - Wed.)
>
>
> 15 - 19 Juni 2009  (E)  3-day qPCR Core Module (Mon. - Wed.) & 2-day
> BioStatistics (Thu. - Fri.)
> 13 - 15 Juli 2009  (E)   NEW microRNA qPCR  (in cooperation with
> Qiagen)
> 27 - 31 Juli 2009  (E)  3-day qPCR Core Module (Mon. - Wed.) & 2-day
> BioStatistics (Thu. - Fri.)
> 14 - 16 September 2009  (E)      NEW  single-cell qPCR
> 19 - 23 September 2009  (E)  3-day microRNA Module (Mon. - Wed.) & 2-
> day BioStatistics (Thu. - Fri.)
> 26 - 28 October 2009 (E)   3-day qPCR Core Module (Mon. - Wed.)
> 16 - 20 November 2009  (E)  3-day microRNA Module (Mon. - Wed.) & 2-
> day BioStatistics (Thu. - Fri.)
> 7 - 11 December 2009  (E)  3-day qPCR Core Module (Mon. - Wed.) & 2-
> day BioStatistics (Thu. - Fri.)
>
> => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf
> => http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf
> => http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pdf
>
>
>
> --------------------------------------------------------------------------------
>
> Forward Please send the qPCR NEWS to further scientists and friends
> who are interested in qPCR !
>
>
> Best regards,
>
> Michael W. Pfaffl
> responsible Editor of the Gene Quantification Pages
> http://www.gene-quantification.info
>
>
>
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>
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>
> ------------------------------
>
> Message: 3
> Date: Thu, 28 May 2009 21:59:16 +0800
> From: "Gourvendu Saxena" <saxena from nus.edu.sg>
> Subject: EDTA solution becomes slurry
> To: <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <D0061E3EA5A8D9458EBAEDBCCA9E9C92057C40FC from MBX21.stu.nus.edu.sg>
> Content-Type: text/plain; charset="iso-8859-1"
>
>
>   Dear All
>   I am trying to prepare 0.5 M EDTA solution pH 8. I used 10 N NaOH for
>   = adjusting the pH but every time it becomes a white semisolid slurry
>   = before reaching the desired pH. Please forward your suggestions.
>   Thanks
>   Gourvendu
>
>
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> End of Methods Digest, Vol 48, Issue 21
> ***************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210


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