Methods Digest, Vol 18, Issue 14

[Virash Gupta]

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:
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> 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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> End of Methods Digest, Vol 18, Issue 14
> ***************************************
>