PAGE purification and analysis (yan sun)

[Manuel Fernando Ariza Botero]

What kind of loading buffer are you using?
Why is the PAGE concentration so high?

Fernando Ariza 

----- Mensaje original -----
De: methods-request from oat.bio.indiana.edu
Fecha: Martes, Abril 8, 2008 12:04 pm
Asunto: Methods Digest, Vol 35, Issue 4

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> Today's Topics:
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>   1. Direct Sequencing of degenerated PCR products (adrien)
>   2. PAGE purification and analysis (yan sun)
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> -------------------------------------------------------------------
> ---
> 
> Message: 1
> Date: Mon, 07 Apr 2008 08:49:16 +0300
> From: adrien <adrien.vetterli from helsinki.fi>
> Subject: Direct Sequencing of degenerated PCR products
> To: methods from magpie.bio.indiana.edu
> Message-ID: <47F9B5DC.5000404 from helsinki.fi>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Hi everybody,
> 
> I would like to sequence PCR products obtained from a reaction 
> with 
> degenerated primers without having to go through the cloning 
> thing. The 
> primers to  be used in the sequencing reaction are degenerated as 
> well. 
> Has anyone tried that who can tell me if the results are usable ? 
> I know 
> of some scientists who have designed degenerated primers with SP6 
> or T7 
> extensions and use these sites during sequencing instead of the 
> degenerated ones but I am not going there either.
> 
> thank you for your help.
> 
> adrien
> 
> 
> ------------------------------
> 
> Message: 2
> Date: Sun, 6 Apr 2008 20:15:14 -0400
> From: "yan sun" <yansun2005 from gmail.com>
> Subject: PAGE purification and analysis
> To: Methods from magpie.bio.indiana.edu
> Message-ID:
> 	<a3be9a9a0804061715v5f123789k7317279d1f236f4b from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
> 
> Dear all,
> I was confused  by my PAGE gel purification for a while.
> I tried to purified spin labeled DNA and non labeled DNA through 20%
> polyacrylamide gel. My DNA is around 8,200 dalton, the attached 
> spin label
> material MW is around 200g/mole, which means I try to separate two DNA
> bands, the molecular weight difference is 200 dalton. My DNA is 27 
> mer.The problem is that two bands are overlapped. the band isn't 
> sharp, the
> resolution is not good, the band is in meniscus shape. wasn't 
> straight at
> all:(
> My lab mate who successfully purified this two band  get very 
> sharp and
> clear bands.I "exactly" follow her protocol. but do not get the 
> satisfiedresult:(
> I used constant power, 15w.
> my guess is the temperature is too high, should I run the gel in 
> the cold
> room? but I pretty sure she run it in RT.
> I pour 25ml acrylamide solution, should I degas it first? my 10% 
> APS is new,
> TEMED is good. is that because the poor gel quality?
> but she didn't degas the gel. I know it really depends on 
> individual hand-on
> experience.
> I don't really know how to improve it, how to get sharp and clear 
> bands?Anybody has suggestion?
> Thx a lot!
> 
> -- 
> Y. F.S.
> 
> 
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