Methods Digest, Vol 18, Issue 16

[Virash Gupta]

answer to Question about protein dialysis-bag leakage (DY)
The protein you are working with might probably be an enzyme with
disolving/ hydrolysing activity on the dialysis bag memebrane. Change
the type of material of bag it may solve the problem.

On 11/17/06, methods-request At oat.bio.indiana.edu
<methods-request At oat.bio.indiana.edu> wrote:
> Send Methods mailing list submissions to
>        methods At net.bio.net
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>
> Today's Topics:
>
>   1. Question from Colombia (Edgar Andres Pulido Bravo)
>   2. Question about protein dialysis-bag leakage (DY)
>   3. troubles with in situ hybridization (Manuel Fernando Ariza Botero)
>   4. Re: Question about protein dialysis-bag leakage (DK)
>   5. UV and the PCR machine? (Nenad Malenica)
>   6. Re: Methods Digest, Vol 18, Issue 15 (Virash Gupta)
>   7. Re: Methods Digest, Vol 18, Issue 14 (Virash Gupta)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 16 Nov 2006 12:03:10 -0500
> From: Edgar Andres Pulido Bravo <eapulidob At unal.edu.co>
> Subject: Question from Colombia
> To: methods At magpie.bio.indiana.edu
> Message-ID: <f1259afe41fc.455c537e At unal.edu.co>
> Content-Type: text/plain; charset="windows-1252"
>
> Drs.
>
> I'm addressing to you about some troubles we are having with the in situ hybridization technique.
>
> Currently we are working with in situ the hybridization to detect Streptococcus agalactiae in tilapia tissues. However, the technique has not been working well, neither on tissues nor on nylon membrane (in spite of using DNA purified from bacteria, as a control positive). We have tried with two probes (length fragment 860 and 190bp) labeling with biotin dCTP incorporated by PCR (on the nylon membrane the probe shows a good labeling). On the slides, with tissues fixed with formalin, we get only a very low signal and just in cerebral tissue.
>
> Attached you can see the protocol we are using. We would like to know if you can give us some comments or recommendations to get better results.
>
> Thanks a lot.
>
> Andres Pulido B.
> National University from Colombia
>
> ------------------------------
>
> Message: 2
> Date: 16 Nov 2006 14:14:25 -0800
> From: "DY" <bearofthecity At gmail.com>
> Subject: Question about protein dialysis-bag leakage
> To: methods At net.bio.net
> Message-ID: <1163715265.519235.192350 At b28g2000cwb.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> I don't know whetehr anyone have encountered the same strange problem.
> I have one protein that is purifified from Ni column. The major peak is
> eluted with about 250 mM immidazole. I tried to dialyze this protein to
> some other buffer. The remarkable thing is once I add protein solution
> into the dialysis bag, the bag starts to leak!
> The bag is not leaky initially, as checked by only adding buffer into
> it. Therefore, the protein definitely plays a role in this leaky issue.
>
> Have anyone seen anything like this? I am really puzzled because this
> seems so counter intuitive.
>
>
>
> ------------------------------
>
> Message: 3
> Date: Thu, 16 Nov 2006 18:20:14 -0500
> From: Manuel Fernando Ariza Botero <mfarizab At unal.edu.co>
> Subject: troubles with in situ hybridization
> To: methods At magpie.bio.indiana.edu
> Cc: Edgar Andres Pulido Bravo <eapulidob At unal.edu.co>
> Message-ID: <f65995cb59f7.455cabde At unal.edu.co>
> Content-Type: text/plain; charset=windows-1252
>
> Drs.
>
> I'm addressing to you about some troubles we are having with the in situ hybridization technique.
>
> Currently we are working with in situ the hybridization to detect Streptococcus agalactiae in tilapia tissues. However, the technique has not been working well, neither on tissues nor on nylon membrane (in spite of using DNA purified from bacteria, as a control positive). We have tried with two probes (length fragment 860 and 190bp) labeling with biotin dCTP incorporated by PCR (on the nylon membrane the probe shows a good labeling). On the slides, with tissues fixed with formalin, we get only a very low signal and just in cerebral tissue.
>
> Attached you can see the protocol we are using. We would like to know if you can give us some comments or recommendations to get better results.
>
> Thanks a lot.
>
> Andres Pulido B.
> National University from Colombia
>
>
>
>
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Fri, 17 Nov 2006 02:03:36 GMT
> From: dk At no.email.thankstospam.net (DK)
> Subject: Re: Question about protein dialysis-bag leakage
> To: methods At net.bio.net
> Message-ID: <YL87h.863$s%7.502 At newsfe02.lga>
>
> In article <1163715265.519235.192350 At b28g2000cwb.googlegroups.com>, "DY" <bearofthecity At gmail.com> wrote:
> >I don't know whetehr anyone have encountered the same strange problem.
> >I have one protein that is purifified from Ni column. The major peak is
> >eluted with about 250 mM immidazole. I tried to dialyze this protein to
> >some other buffer. The remarkable thing is once I add protein solution
> >into the dialysis bag, the bag starts to leak!
> >The bag is not leaky initially, as checked by only adding buffer into
> >it. Therefore, the protein definitely plays a role in this leaky issue.
> >
> >Have anyone seen anything like this? I am really puzzled because this
> >seems so counter intuitive.
>
> Wow. Where does the leak happen? How do you close the bag's ends?
>
> If it's a chemical thing (hard to believe!) then you can try less
> conventional membranes made from cellulose ester or even PVDF
> type.
>
> DK
>
>
>
> ------------------------------
>
> Message: 5
> Date: Fri, 17 Nov 2006 08:20:22 +0100
> From: Nenad Malenica <malenica At biol.pmf.hr>
> Subject: UV and the PCR machine?
> To: methods At magpie.bio.indiana.edu
> Message-ID: <455D62B6.2040602 At zg.biol.pmf.hr>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hello,
> Thanks for the suggestions. That will help. To answer the question: I
> want to do PCR with archaeological and contemporary samples. Since we
> have only one PCR machine I have not much choice. I know that for
> a(ncient)DNA  I would need a separate lab, but I hope that with proper
> controls I might get the information I need.
> Regards,
> Nenad
>
>
>
> ------------------------------
>
> Message: 6
> Date: Fri, 17 Nov 2006 10:02:54 +0530
> From: "Virash Gupta" <virashkgupta At gmail.com>
> Subject: Re: Methods Digest, Vol 18, Issue 15
> To: methods At oat.bio.indiana.edu
> Message-ID:
>        <f5d8ce8c0611162032q4179dba3re87a3da42ded9682 At mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> No need to decontaminate as PCR reaction mix in closed tubes can not
> contaminate the PCR block except for when PCR tubes are wrongly
> handeled during reaction setup. Some PCR machines may loose efficiency
> due deposition of dust or dirt with time. In such a case, for to be
> sure of cleanlness, wipe inside of PCR block holes with a mild
> detergent sol, let it stand for 5-10 min and then wipe with clean
> water and 70 % Etanol followed bydrying with kimwipe or other nonfibre
> relasing paper. You need to do it manually using cotton swab made on a
> metal wire.
> V K Gupta
>
> On 11/16/06, methods-request At oat.bio.indiana.edu
> <methods-request At oat.bio.indiana.edu> wrote:
> > Send Methods mailing list submissions to
> >        methods At net.bio.net
> >
> > To subscribe or unsubscribe via the World Wide Web, visit
> >        http://www.bio.net/biomail/listinfo/methods
> > or, via email, send a message with subject or body 'help' to
> >        methods-request At net.bio.net
> >
> > You can reach the person managing the list at
> >        methods-owner At net.bio.net
> >
> > When replying, please edit your Subject line so it is more specific
> > than "Re: Contents of Methods digest..."
> >
> >
> > Today's Topics:
> >
> >   1. Re: UV and the PCR machine? (Jose de las Heras)
> >   2. Re: UV and the PCR machine? (DK)
> >
> >
> > ----------------------------------------------------------------------
> >
> > Message: 1
> > Date: Thu, 16 Nov 2006 00:33:02 -0000
> > From: "Jose de las Heras" <josenet At tiscali.co.uk>
> > Subject: Re: UV and the PCR machine?
> > To: methods At net.bio.net
> > Message-ID: <4s1pv0Ft46iuU1 At mid.individual.net>
> >
> >
> > "Nenad Malenica" <malenica At biol.pmf.hr> wrote in message
> > news:mailman.37.1163602169.19683.methods At net.bio.net...
> > > Hello there,
> > > I plan to "decontaminate" my  PCR machine (the block) by  exposing it to
> > > UV overnight (in the laminar I have a 30W UV lamp). But I am not sure if I
> > > can harm the machine in any way by this treatment.  I suppose HCl or NaOCl
> > > (bleach) is out of the question (to clean the block itself). Any
> > > suggestions?
> > > Nenad
> >
> > why do you want to do that?
> >
> > the reactions are mixed and the tubes closed by the time they come near the
> > machine. I'd have assumed the chances of contamination sourced to the
> > machine would be practically non-existent.
> >
> > Jose
> >
> >
> >
> >
> > ------------------------------
> >
> > Message: 2
> > Date: Thu, 16 Nov 2006 02:40:25 GMT
> > From: dk At no.email.thankstospam.net (DK)
> > Subject: Re: UV and the PCR machine?
> > To: methods At net.bio.net
> > Message-ID: <scQ6h.47$6e4.21 At newsfe02.lga>
> >
> > In article <4s1pv0Ft46iuU1 At mid.individual.net>, "Jose de las Heras" <josenet At tiscali.co.uk> wrote:
> > >
> > >"Nenad Malenica" <malenica At biol.pmf.hr> wrote in message
> > >news:mailman.37.1163602169.19683.methods At net.bio.net...
> > >> Hello there,
> > >> I plan to "decontaminate" my  PCR machine (the block) by  exposing it to
> > >> UV overnight (in the laminar I have a 30W UV lamp). But I am not sure if I
> > >> can harm the machine in any way by this treatment.  I suppose HCl or NaOCl
> > >> (bleach) is out of the question (to clean the block itself). Any
> > >> suggestions?
> > >> Nenad
> > >
> > >why do you want to do that?
> > >
> > >the reactions are mixed and the tubes closed by the time they come near the
> > >machine. I'd have assumed the chances of contamination sourced to the
> > >machine would be practically non-existent.
> >
> > Seconded.
> >
> >
> >
> > ------------------------------
> >
> > _______________________________________________
> > Methods mailing list
> > Methods At net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
> > End of Methods Digest, Vol 18, Issue 15
> > ***************************************
> >
>
>
>
> ------------------------------
>
> Message: 7
> Date: Fri, 17 Nov 2006 10:12:17 +0530
> From: "Virash Gupta" <virashkgupta At gmail.com>
> Subject: Re: Methods Digest, Vol 18, Issue 14
> To: methods At oat.bio.indiana.edu
> Message-ID:
>        <f5d8ce8c0611162042h16a4fbfeo78371e876f6834df At mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> why you want to decontaminate the machine, from DNA or bacterial
> contaminants or dust/dirt. DNA need to be brocken either with UV or
> wiping with 0.1N HCl solfollowed by with 70% ethanol and water using a
> swab.Bleach will kill bacterial bacteria but will not denatureor
> degrade DNa contamination if their is any.
>
> On 11/15/06, methods-request At oat.bio.indiana.edu
> <methods-request At oat.bio.indiana.edu> wrote:
> > Send Methods mailing list submissions to
> >        methods At net.bio.net
> >
> > To subscribe or unsubscribe via the World Wide Web, visit
> >        http://www.bio.net/biomail/listinfo/methods
> > or, via email, send a message with subject or body 'help' to
> >        methods-request At net.bio.net
> >
> > You can reach the person managing the list at
> >        methods-owner At net.bio.net
> >
> > When replying, please edit your Subject line so it is more specific
> > than "Re: Contents of Methods digest..."
> >
> >
> > Today's Topics:
> >
> >   1. Re: PCR band smear (Duncan Clark)
> >   2. Re: PCR band smear (Domo)
> >   3. smearing in PCR amplification run (Virash Gupta)
> >   4. siRNA transfection of primary neurons (O'Hare)
> >   5. DEPC inactivation (Hemert, M.J. van (MM))
> >   6. Re: PCR band smear (Damien)
> >   7. 5' labelling with S-35 (Khelifa Arab)
> >   8. Re: PCR band smear (Duncan Clark)
> >   9. UV and the PCR machine? (Nenad Malenica)
> >  10. Re: UV and the PCR machine? (jg374 At cam.ac.uk)
> >  11. Re: UV and the PCR machine? (WS)
> >
> >
> > ----------------------------------------------------------------------
> >
> > Message: 1
> > Date: Tue, 14 Nov 2006 12:47:24 +0000
> > From: Duncan Clark <blackhole At abuse.plus.com>
> > Subject: Re: PCR band smear
> > To: methods At net.bio.net
> > Message-ID: <Qt3cx9BcrbWFFA$T At abuse.plus.com>
> > Content-Type: text/plain;charset=us-ascii;format=flowed
> >
> > Historians believe that in newspost
> > <mailman.8.1163443809.19683.methods At net.bio.net> on Sun, 12 Nov 2006,
> > muhammad yasir <yasirphr At yahoo.com> penned the following literary
> > masterpiece:
> > >i am running PCR unsing PCR product as templet and facing the problem of smear at extactly the same position aroung of desired band size. Any
> > >sugestion for this problem
> >
> > How much are you diluting the original PCR product and how many cycles
> > are you then doing ?
> >
> > Duncan
> > --
> > I love deadlines. I especially like the whooshing noise they make as
> > they go flying by.
> >
> > Duncan Clark
> > GeneSys Ltd.
> >
> >
> > ------------------------------
> >
> > Message: 2
> > Date: Tue, 14 Nov 2006 19:37:10 +0800
> > From: "Domo" <rimask At yahoo.com>
> > Subject: Re: PCR band smear
> > To: methods At net.bio.net
> > Message-ID: <4559aa63$1 At news.starhub.net.sg>
> >
> > Ta ==> 60C for 30 sec
> >
> > increase annealing temperature
> >
> > "muhammad yasir" <yasirphr At yahoo.com> wrote in message
> > news:mailman.8.1163443809.19683.methods At net.bio.net...
> > >i am running PCR unsing PCR product as templet and facing the problem of
> > >smear at extactly the same position aroung of desired band size. Any
> > >sugestion for this problem
> > >  with thanks
> > >  Yasir
> > >
> > >
> > > ---------------------------------
> > > Want to start your own business? Learn how on Yahoo! Small Business.
> >
> >
> >
> >
> > ------------------------------
> >
> > Message: 3
> > Date: Tue, 14 Nov 2006 22:56:14 +0530
> > From: "Virash Gupta" <virashkgupta At gmail.com>
> > Subject: smearing in PCR amplification run
> > To: methods At magpie.bio.indiana.edu
> > Message-ID:
> >        <f5d8ce8c0611140926gfd86834h1688472187a10274 At mail.gmail.com>
> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> >
> > Dear zarrin eshaghi,
> > I have faced this problem occasionally both using DNA template and PCR
> > product as template. More often this is due to high protein content in
> > the DNA product to be run. The proteins are contributed by poor
> > quality of taq, poor quality of template DNA and sometimes due to high
> > salt concentration (MgCl2, template DNA). In your case reamplifying
> > the PCR product did not solve the problem. It seems to be related to
> > poor quality Taq ( from supplier or home made taq). Try to change the
> > source of Taq and problem is likely to be solved. Also try using lower
> > amount of taq per reation as taq itself being protein contributes to
> > proteins. good luck.
> >
> > Dr V K Gupta
> > Insect Molecular Biology lab
> > Dept of Entomology
> > Punjab Agricultural University
> > Ludhiana-Pb-141004-India
> > virashkgupta At gmail.com
> >
> >
> >
> > ------------------------------
> >
> > Message: 4
> > Date: Tue, 14 Nov 2006 12:40:11 -0500
> > From: O'Hare, Michael<Michael.OHare At nrc-cnrc.gc.ca>
> > Subject: siRNA transfection of primary neurons
> > To: <neur-sci At magpie.bio.indiana.edu>,
> >        <methods At magpie.bio.indiana.edu>
> > Message-ID:
> >        <F1F7560D7EB35B40B78E9EB14CB2992001D62351 At nrccenexb2.nrc.ca>
> > Content-Type: text/plain;       charset="us-ascii"
> >
> > Has anybody managed to successfully transfect primary cultures of
> > cerebellar granule neurons with short double stranded siRNA?  If so,
> > what transfection reagent did you use and how much RNA and transfection
> > reagent needs to be combined to achieve optimal transfection?  Any
> > additional details that could be provided would also be greatly
> > appreciated.  Thank you.
> >
> >
> >
> > -------------------------------------------------------------------
> >
> > Michael J. O'Hare, Ph.D.
> >
> > Tel: (613) 993-4294 | Fax: (613) 941-4475 | email: michael.ohare At nrc.ca
> >
> >
> >
> > Institute for Biological Sciences
> >
> > National Research Council of Canada
> >
> > 1200 Montreal Road, Building M-54
> >
> > Ottawa, Ontario K1A 0R6, Canada
> >
> > -------------------------------------------------------------------
> >
> >
> >
> >
> >
> > ------------------------------
> >
> > Message: 5
> > Date: Tue, 14 Nov 2006 16:43:16 +0100
> > From: "Hemert, M.J. van \(MM\)" <M.J.van_Hemert At lumc.nl>
> > Subject: DEPC inactivation
> > To: <methods At magpie.bio.indiana.edu>
> > Message-ID:
> >        <F61D0762A9772840A574C78F6D33C9D48AD2AF At mailb.lumcnet.prod.intern>
> > Content-Type: text/plain;       charset="us-ascii"
> >
> > TRIS reacts with and inactivates your DEPC
> >
> >
> > ------------------------------
> >
> > Message: 6
> > Date: Tue, 14 Nov 2006 13:19:52 -0600
> > From: Damien <marsicd At removethis.uah.edu>
> > Subject: Re: PCR band smear
> > To: methods At net.bio.net
> > Message-ID: <ejd4sn$ng3$1 At news.uah.edu>
> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> >
> > You can reduce smearing by using less DNA polymerase, decreasing the
> > number of cycles, or decreasing extension time. Increasing template
> > concentration might help as well.
> >
> > You may also try another DNA polymerase. I use Rainbow
> > (www.extremozyme.com).
> >
> > Damien
> >
> >
> >
> > muhammad yasir wrote:
> > > i am running PCR unsing PCR product as templet and facing the problem of smear at extactly the same position aroung of desired band size. Any sugestion for this problem
> > >   with thanks
> > >   Yasir
> > >
> > >
> > > ---------------------------------
> > > Want to start your own business? Learn how on Yahoo! Small Business.
> >
> >
> > ------------------------------
> >
> > Message: 7
> > Date: Wed, 15 Nov 2006 10:25:42 +0100
> > From: "Khelifa Arab" <a.khelifa At dkfz-heidelberg.de>
> > Subject: 5' labelling with S-35
> > To: <methods At magpie.bio.indiana.edu>
> > Message-ID:
> >        <F30745375DA03E45A233B025D152FB35012E00E9 At dkfzex1.ad.dkfz-heidelberg.de>
> >
> > Content-Type: text/plain;       charset="us-ascii"
> >
> > Hello,
> >
> >
> >
> > I find your email address in the discussion of T4PNK labeling with
> > ATPgS35. Could you please provide me informations about the reaction of
> > T4PNK with single nucleosides in the presence of ATPgS35.
> >
> > Sincerely,
> >
> > Khelifa Arab
> >
> >
> >
> > Division of Toxicology and Cancer Risk Factors
> > German Cancer Research Center (DKFZ)
> > Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
> > Phone +49 6221-423304 (office)
> > Phone +49 6221-423322 (lab)
> >
> > Fax +49 6221-423359
> > a.khelifa At dkfz.de
> >
> >
> >
> >
> >
> > ------------------------------
> >
> > Message: 8
> > Date: Wed, 15 Nov 2006 12:41:00 +0000
> > From: Duncan Clark <blackhole At abuse.plus.com>
> > Subject: Re: PCR band smear
> > To: methods At net.bio.net
> > Message-ID: <DGHo7xAcrwWFFAO7 At abuse.plus.com>
> > Content-Type: text/plain;charset=us-ascii;format=flowed
> >
> > Historians believe that in newspost <ejd4sn$ng3$1 At news.uah.edu> on Tue,
> > 14 Nov 2006, Damien <marsicd At removethis.uah.edu> penned the following
> > literary masterpiece:
> > >You may also try another DNA polymerase. I use Rainbow
> > >(www.extremozyme.com).
> >
> > Ah, self advertising.
> >
> > Duncan
> > --
> > I love deadlines. I especially like the whooshing noise they make as
> > they go flying by.
> >
> > Duncan Clark
> > GeneSys Ltd.
> >
> >
> > ------------------------------
> >
> > Message: 9
> > Date: Wed, 15 Nov 2006 15:12:18 +0100
> > From: Nenad Malenica <malenica At biol.pmf.hr>
> > Subject: UV and the PCR machine?
> > To: "methods At net.bio.net" <methods At magpie.bio.indiana.edu>
> > Message-ID: <455B2042.80602 At zg.biol.pmf.hr>
> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> >
> > Hello there,
> > I plan to "decontaminate" my  PCR machine (the block) by  exposing it to
> > UV overnight (in the laminar I have a 30W UV lamp). But I am not sure if
> > I can harm the machine in any way by this treatment.  I suppose HCl or
> > NaOCl (bleach) is out of the question (to clean the block itself). Any
> > suggestions?
> > Nenad
> >
> >
> >
> > ------------------------------
> >
> > Message: 10
> > Date: Wed, 15 Nov 2006 14:56:36 +0000
> > From: "jg374 At cam.ac.uk" <jg374 At hermes.cam.ac.uk>
> > Subject: Re: UV and the PCR machine?
> > To: methods At net.bio.net
> > Message-ID: <Pine.LNX.4.64.0611151455040.23181 At hermes-1.csi.cam.ac.uk>
> > Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed
> >
> > On Wed, 15 Nov 2006, Nenad Malenica wrote:
> > > Hello there,
> > > I plan to "decontaminate" my  PCR machine (the block) by  exposing it to UV
> > > overnight (in the laminar I have a 30W UV lamp). But I am not sure if I can
> > > harm the machine in any way by this treatment.  I suppose HCl or NaOCl
> > > (bleach) is out of the question (to clean the block itself). Any suggestions?
> > > Nenad
> >
> > A few companies sell products designed for this such as DNA Away or
> > Nucleoclean.
> >
> >
> > ------------------------------
> >
> > Message: 11
> > Date: 15 Nov 2006 07:44:16 -0800
> > From: "WS" <novalidaddress At nurfuerspam.de>
> > Subject: Re: UV and the PCR machine?
> > To: methods At net.bio.net
> > Message-ID: <1163605456.237558.188670 At h48g2000cwc.googlegroups.com>
> > Content-Type: text/plain; charset="us-ascii"
> >
> > Dear Nenad,
> >
> > UV may damage plastics and LCD-displays. If you just want to irradiate
> > the block, cover the rest with paper etc. Overnight might be overkill.
> > 30 min at a short distance should be sufficient. Do you think, some DNA
> > gets from the block into your tubes? If your block is made from
> > stainless steel, then bleach is fine. However, it might damage parts
> > made of aluminium and almost everything that gets spilled with bleach
> > due it's alkaline.
> >
> > Best regards, Wo
> >
> >
> > Nenad Malenica wrote:
> > > Hello there,
> > > I plan to "decontaminate" my  PCR machine (the block) by  exposing it to
> > > UV overnight (in the laminar I have a 30W UV lamp). But I am not sure if
> > > I can harm the machine in any way by this treatment.  I suppose HCl or
> > > NaOCl (bleach) is out of the question (to clean the block itself). Any
> > > suggestions?
> > > Nenad
> >
> >
> >
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