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:
>> 1. Direct Sequencing of degenerated PCR products (adrien)
> 2. PAGE purification and analysis (yan sun)
>>> -------------------------------------------------------------------
> ---
>> 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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