Viability of Bacterial culture at -4C

Siddhartha Parida sidharth4 at gmail.com
Fri Mar 31 00:20:11 EST 2006


Will bacterial culture stored at 30% glycerol stock be viable for 6
months to 1 yr at -4C , Generally it is mentioned to keep it at -80C

On 3/31/06, methods-request at oat.bio.indiana.edu
<methods-request at oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. separation of single and double strand RNA/DNA comlpex (ali)
>   2. Re: PCR cleanup before digest (Cornelius Fritsch)
>   3. RE: Annealing Temperatures for PCR (Deanne Bell)
>   4. sequence variation (as)
>   5. Re: pH neutralisation of samples for SDS-PAGE (Nick Theodorakis)
>   6. looking to buy spectrophotomer (prabhu ram)
>   7. subscriber problem (Luzan Tatiana)
>   8. Re: DNA from old frozen and partly coagulated heparin blood
>      (marc crepeau)
>   9. Re: Re-POST of THE ORIGINAL MERLIN MINI/MAXI-PREP
>      (Wolfgang Schechinger)
>  10. RE:  (Jayakumar, R)
>  11. RE: PCR cleanup before digest (Jayakumar, R)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: 30 Mar 2006 08:06:30 -0800
> From: "ali" <atahery at gmail.com>
> Subject: separation of single and double strand RNA/DNA comlpex
> To: methods at net.bio.net
> Message-ID: <1143734789.986212.103550 at t31g2000cwb.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Is there any method to separate single and double stranded RNA/DNA
> comples from each other(Other than hydroxyapatite columns)
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 30 Mar 2006 19:36:02 +0200
> From: "Cornelius Fritsch" <C.Fritsch at dkfz-heidelberg.de>
> Subject: Re: PCR cleanup before digest
> To: methods at net.bio.net
> Message-ID: <op.s68luchoy4bjlh at anjacorni>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> On Thu, 30 Mar 2006 10:26:37 +0200, newsnet customer
> <customer at newsnet.com> wrote:
>
> > How does proof-reading enzymes affect downstream cloning?
> >
> > ST
> >
> They can "proof-read" away your (unpaired, 3'-exposed) overhangs.
>
> Cornelius
>
>
> ------------------------------
>
> Message: 3
> Date: Thu, 30 Mar 2006 08:01:37 -0800
> From: "Deanne Bell" <dbell at fresno.ars.usda.gov>
> Subject: RE: Annealing Temperatures for PCR
> To: "Methods Forum new address" <methods at magpie.bio.indiana.edu>
> Message-ID:
>        <D7450475FF8D7B4DAE55A71352AACFB705A70C at exchange.fresno.ars.usda.gov>
> Content-Type: text/plain;       charset="us-ascii"
>
> I totally agree with Jai - you really need to run your sequences thru a
> calculator to get a GENERAL idea and then run PCRs with a range of
> annealing tempuratures (I LOVE my gradient thermocycler).  Then you will
> want to optimize the amount of Mg++ and the amount of DNA added for your
> specific type of samples.
>
>
> For example:
> * plant DNA seems to prefer ~4mM MgCl, while the bacteria I work with
> amplifies best with 1.5mM.
> * some DNA extraction protocols will leave left-over substances that are
> inhibitory to PCR thus less of the DNA prep must be used to get good
> amplification (this is really counter-intuitive when you see light bands
> being produced).  Too much DNA will throw off the PCR reaction also.
>
> Good luck, hope this helps
> Deanne Bell
>
>
>
>
>
> >-----Original Message-----
> >From: methods-bounces at oat.bio.indiana.edu
> >[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Jayakumar, R
> >Sent: Thursday, March 30, 2006 6:35 AM
> >To: methods at magpie.bio.indiana.edu
> >Subject: RE: Annealing Temperatures for PCR
> >
> > You can calculate it using several online tools. One of my
> >favourite is
> >   http://www.basic.northwestern.edu/biotools/oligocalc.html
> >You can also search on google with keywords "annealing
> >temperature calculator tool".  The melting temperature is your
> >annealing temperature.
> >
> >   But still the actual Annealing temperature has tto be
> >optimized through gradient PCR using different conc. Off Mg
> >and salts.  The Tm values only helps to give an idea.
> >Best of luck
> >Jayakumar
> >
> >-----Original Message-----
> >From: methods-bounces at oat.bio.indiana.edu
> >[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of
> >retnohh at mail.mitra.net.id
> >Sent: Wednesday, March 29, 2006 6:31 PM
> >To: methods at magpie.bio.indiana.edu
> >Subject: Annealing Temperatures for PCR
> >
> >Dear sir,
> > From internet I know that youa an expert in Molecular Biology
> >research.
> >I have a problem with my student PCR.
> >If you do not mind, please help me to tell me how to calculate
> >the exact annealing primers for pair of primers:
> >5'- TTA GGG CAA GAG ATG GTA AGG -3'  and
> >5'- TTA TAA CAA TGA TGG AGG G -3'
> >Thank you very much for your kindness.
> >
> >Best regard
> >Retnohh
> >
> >
> >_______________________________________________
> >Methods mailing list
> >Methods at net.bio.net
> >http://www.bio.net/biomail/listinfo/methods
> >
> >
> >This email message may contain legally privileged and/or
> >confidential information.  If you are not the intended
> >recipient(s), or the employee or agent responsible for the
> >delivery of this message to the intended recipient(s), you are
> >hereby notified that any disclosure, copying, distribution, or
> >use of this email message is prohibited.  If you have received
> >this message in error, please notify the sender immediately by
> >e-mail and delete this email message from your computer. Thank you.
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>
>
>
> ------------------------------
>
> Message: 4
> Date: Thu, 30 Mar 2006 18:43:41 +0200
> From: "as" <nospam at nospamss.dk>
> Subject: sequence variation
> To: methods at net.bio.net
> Message-ID: <4VTWf.99$8I3.95 at news.get2net.dk>
>
> Hi NG.
> I seem to remember from my genetic class that one way of generating sequence
> variation in genes (or gene families) is that transcribed mRNA recombines
> back into the genome, and as a consequence of the lower fidelity of the RNA
> pol there will be point mutations in some of the mRNAs! Could anyone please
> provide me with a reference on this phenonemon.
> Thanks in advance
> AS
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: 30 Mar 2006 09:42:01 -0800
> From: "Nick Theodorakis" <nick_theodorakis at hotmail.com>
> Subject: Re: pH neutralisation of samples for SDS-PAGE
> To: methods at net.bio.net
> Message-ID: <1143740521.411017.47620 at i39g2000cwa.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
>
> Amanda Gillespie wrote:
> > I have neutrophil homogenate samples, which I am treating with a glycanase
> > to deglycosylate the protein I am interested in, to be followed by reverse
> > zymography to test the activity of this protein.
> >
> > Any suggestions/opinions/comments on how to neutralise the pH after
> > deglycosylation, as the buffer required for this is very low -
> > 5.0citrate-phosphate, which is considerably lower than the optimal pH
> > for my
> > protein, and inteferes with the activity shown on the reverse zymogram...
> >
> > I tried just adding normal non-reducing treatment buffer to my samples after
> > deglycosylation, hoping that would be enough to adjust the pH, but
> > apparently, it isn't :(
>
> Assuming you are using bromophenol blue as the indicator dye-- make a
> small stock of unbuffered saturated Tris base. Titrate in small amounts
> (e.g., 1 or 2 microliters or so) of base into your sample until the dye
> turns from yellow/orange back to blue.
>
> Nick
>
> --
> Nick Theodorakis
> nick_theodorakis at hotmail.com
> contact form:
> http://theodorakis.net/contact.html
>
>
>
> ------------------------------
>
> Message: 6
> Date: Thu, 30 Mar 2006 20:43:27 +0200
> From: "prabhu ram" <prabhu at mriegypt.com>
> Subject: looking to buy spectrophotomer
> To: <methods at magpie.bio.indiana.edu>
> Message-ID: <C2FD72990F335443822090D1639F30C505906A at main.mriegypt.com>
> Content-Type: text/plain;       charset="us-ascii"
>
> Dear Sir,
>
>
>
> We are from Mansoura Resins and chemicals company Egypt.
>
>
>
> We are looking to buy Spectro photomer Latest Model for checking
> chloride content in our Product.
>
>
>
> Please send details of spectrophotomer types.
>
>
>
> Waiting for your reply.
>
>
>
> Regards
>
>
>
> R.A.Praburam
>
>
>
> Quality Control.
>
>
>
>
>
>
>
> ------------------------------
>
> Message: 7
> Date: Thu, 30 Mar 2006 12:44:49 -0600
> From: "Luzan Tatiana" <txl8326 at louisiana.edu>
> Subject: subscriber problem
> To: methods at magpie.bio.indiana.edu
> Message-ID: <20060330184405.M38187 at louisiana.edu>
> Content-Type: text/plain;       charset=iso-8859-1
>
>
> Hey
> How can  I get any advice about the anaer anaerobic media preparation?
> let me know, how can I post my message on the forum
> --
>
>  Tanya Luzan
> Research Assistant
> Department of Biology
> University of Louisiana at Lafayette
> Phone (Office): 482-5056
>
>
>
> ------------------------------
>
> Message: 8
> Date: 30 Mar 2006 11:28:58 -0800
> From: "marc crepeau" <mwcrepeau at hawaii.rr.com>
> Subject: Re: DNA from old frozen and partly coagulated heparin blood
> To: methods at net.bio.net
> Message-ID: <1143746938.481099.192500 at i39g2000cwa.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> I'd be curious to know how you determined that there was no DNA.
> Agarose gel?  Spectrophotometry?  Also, are you using PCR based methods
> for your genetic analysis?  In my experience, DNA from
> heparin-preserved whole blood is useless for PCR since the heparin
> inhibits the reaction and is impossible to remove.  But Novagen sells a
> product called BloodDirect that they claim allows PCR directly from
> whole blood stored fresh or frozen with a variety of anti-coagulants,
> including heparin.  I've never tried it, but you might want to look
> into it.  Good luck!
>
> -Marc
>
>
>
> ------------------------------
>
> Message: 9
> Date: 30 Mar 2006 13:03:55 -0800
> From: "Wolfgang Schechinger" <novalidaddress at nurfuerspam.de>
> Subject: Re: Re-POST of THE ORIGINAL MERLIN MINI/MAXI-PREP
> To: methods at net.bio.net
> Message-ID: <1143752635.782491.152660 at j33g2000cwa.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Reads great.
>
> I just wanted to add that, for most purposes, the Maniatis-Style
> minipreps are even simplier:
>
> Grow bacteria in LB in an eppi with some holes punched in the lid
> Pellet bacteria by 5min at 5000g (approx)
> add 150 ul of suspension buffer (you may use Merlin 1), scratch the
> tube over an eppi stand for shockwave-redistribution, put on ice.
> Add 150ul of Lysis Buffer (0.1M NaOH, 0.2% SDS, you may use Merlin 2),
> scratch agin a few times, put on ice again.
> Add 150ul of potassium acetate/acetic acid neutralization buffer (you
> may use Merlin 3). Scratch again, spin 10-15 min at max. speed.
> In order to precipitate the DNA, add the S/N to 500ul of isopropanol,
> mix, spin for 15 min at max speed, remove S/N, wash again with 500 ul
> of 70% ethanol which is stored at -20 in the dark. Spin 1 min at max
> speed, remove S/N and allow to dry at RT.
>
> I learned of this scratching method by hand_to_hand propaganda.
>
> This protocol yields DNA suitable for digests, PCR and even sequencing
> and transfections (at least for test purposes) at almost no cost and
> shorter hands-on-time than almost any kit. Scale up to 5ml (to be grown
> directly in 15 ml plastic tubes, precipitation is done in one or more
> eppis) and more ml preps (you may need to increase buffer solutions to
> 300ul and morel) is possible.
>
> I just wonder if the MMM (Magic Merlin Method) is used in these nice
> {PCR, digest, etc} cleanup kits where you add binding buffer to your
> sample, apply the mix on some white matter and wash with a wash buffer
> where you have to add ethanol before use and then you elute with water
> or TE.
>
> So I wonder, too, if one might re-use these nice cleanup columns after
> elution?
>
> Anyone tried that yet?
>
> Best regards,
>
> Wolfgang Schechinger
>
> Endocrine Research Lab,
> BG & University Hospital Bergmannsheil
> Bochum
> Germany
>
>
>
> ------------------------------
>
> Message: 10
> Date: Thu, 30 Mar 2006 16:53:09 -0500
> From: "Jayakumar, R" <R.Jayakumar at roswellpark.org>
> Subject: RE:
> To: "vimaleswaran santhanakrishnan" <vinusa15 at yahoo.com>,
>        <methods at magpie.bio.indiana.edu>
> Message-ID:
>        <97101976F8A044468CA74FE11883B90E0749662B at VISTA.roswellpark.org>
> Content-Type: text/plain;       charset="us-ascii"
>
> Yes.. You are right.  But unfortunately, all tools only calculate the Tm
> since annealing temperatures are very much dependent on the PCR machine,
> purity of your DNA, buffers etc etc.. The Tm value will give you an idea
> of the annealing temperature which is generally 5 degrees lower than Tm.
> I am sorry that I did not elaborate on that.
>    Generally, I start my optimization PCRs (with my buddy gradient
> thermocycler) by taking an annealing temperature that is 5 degrees lower
> than caculated melting temperature(Tm) and work  5 degrees both ways in
> my first gradient PCR.  For eg. If the Tm is 65 C, take a gradient
> between 55-65. I also try out 1 - 2.5 mM Mg conc. As well as 2-3
> different template concentrations.  All 3 variables can be checked on a
> single PCR reaction setup in a gradient thermocycler changing only one
> variable in each reaction.  If cDNA is used, use 1 ul of cDNA as
> template (from 25 ul of cDNA from 1ug of RNA).  If total DNA is used,
> start of with 50-100 ng of DNA.  Plasmids require very less anywhere
> from 10 ng to 25 ng should be more than sufficient.
>  Best of luck
> Jai
>
> -----Original Message-----
> From: vimaleswaran santhanakrishnan [mailto:vinusa15 at yahoo.com]
> Sent: Thursday, March 30, 2006 10:31 AM
> To: Jayakumar, R
> Subject:
>
> hello jayakumar,
>      melting temp. cannot be annealing temperature.
> vimal
>
>
>
> --- "Jayakumar, R" <R.Jayakumar at roswellpark.org>
> wrote:
>
> >  You can calculate it using several online tools.
> > One of my favourite is
> >
> ttp://www.basic.northwestern.edu/biotools/oligocalc.html
> > You can also search on google with keywords "annealing temperature
> > calculator tool".  The melting temperature is your annealing
> > temperature.
> >
> >    But still the actual Annealing temperature has tto be optimized
> > through gradient PCR using different conc. Off Mg and salts.  The Tm
> > values only helps to give an idea.
> > Best of luck
> > Jayakumar
> >
> > -----Original Message-----
> > From: methods-bounces at oat.bio.indiana.edu
> > [mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of
> > retnohh at mail.mitra.net.id
> > Sent: Wednesday, March 29, 2006 6:31 PM
> > To: methods at magpie.bio.indiana.edu
> > Subject: Annealing Temperatures for PCR
> >
> > Dear sir,
> >  From internet I know that youa an expert in Molecular Biology
> > research.
> > I have a problem with my student PCR.
> > If you do not mind, please help me to tell me how to calculate the
> > exact annealing primers for pair of primers:
> > 5'- TTA GGG CAA GAG ATG GTA AGG -3'  and
> > 5'- TTA TAA CAA TGA TGG AGG G -3'
> > Thank you very much for your kindness.
> >
> > Best regard
> > Retnohh
> >
> >
> > _______________________________________________
> > Methods mailing list
> > Methods at net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
> >
> > This email message may contain legally privileged and/or confidential
> > information.  If you are not the intended recipient(s), or the
> > employee or agent responsible for the delivery of this message to the
> > intended recipient(s), you are hereby notified that any disclosure,
> > copying, distribution, or use of this email message is prohibited.  If
>
> > you have received this message in error, please notify the sender
> > immediately by e-mail and delete this email message from your
> > computer. Thank you.
> >
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>
>
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> This email message may contain legally privileged and/or confidential information.  If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited.  If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.
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>
>
> ------------------------------
>
> Message: 11
> Date: Thu, 30 Mar 2006 16:55:01 -0500
> From: "Jayakumar, R" <R.Jayakumar at roswellpark.org>
> Subject: RE: PCR cleanup before digest
> To: "newsnet customer" <customer at newsnet.com>,
>        <methods at magpie.bio.indiana.edu>
> Message-ID:
>        <97101976F8A044468CA74FE11883B90E0749662C at VISTA.roswellpark.org>
> Content-Type: text/plain;       charset="us-ascii"
>
> That is new info.  I never heard anything like that.  Anyway, I use only
> 2 ul of PCR product for my digestion and I guess when I make it up to 50
> ul Mg is diluted out.  I get perfect digestions.
>   Best ofluck
> Jai
>
>
> -----Original Message-----
> From: methods-bounces at oat.bio.indiana.edu
> [mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of newsnet
> customer
> Sent: Thursday, March 30, 2006 12:48 AM
> To: methods at magpie.bio.indiana.edu
> Subject: PCR cleanup before digest
>
> Hi,
>
> Been told to do a PCR cleanup before doing a restriction digest because
> the salt (MgCl2) in the PCR mix can affect cleavage efficiency. Anyone
> like to comment?
>
>
> ST
> _______________________________________________
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> This email message may contain legally privileged and/or confidential information.  If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited.  If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.
>
>
>
> ------------------------------
>
> _______________________________________________
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> End of Methods Digest, Vol 10, Issue 30
> ***************************************
>


--
Sidharth Parida
Senior Faculty
Center for biotechnology and Research
Neelachal Inst of Medical Sciences
O.C.H.C building , Near Ram Mandir
Bhubaneswar-3
ORISSA
09437089337(M)
sidharth4 at gmail.com



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