Methods Digest, Vol 56, Issue 15-seperating 3.5 and 3.3 kb in agarosegel

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Wed Jan 20 20:32:16 EST 2010


Seperating 3.5 and 3.3 kb in agarosegel.
It is possible but with patience only by sequential separation on 0.7
% agarose. Run a higher quantity of mixed DNA in a small sized well.
let it run untill it has moved ~75% of the gel. Cut the now slightly
separated mixed band and reload the block keeping care not to change
the direction of cut gel block. This you can do by cutting a gel block
at well site of size slightly bigger than the size of DNA gel block.
Fit the block ( mind direction, the smaller band towards running side.
Fill the side gaps with liquified 0.7% agarose. Let it solidify and
run. Keep track of movement till again 75% of agarose gel has been
run. You will find separation. You can re run or if satisfied with
separartion you can cut  a small block of agarose from outer end of
the band and purify DNA. Alternatively you can clone the mixture into
suitable vector and check the size of the cloned insert. To clone Just
incubate your DNa in PCR reaction mixture using normal aq and only
dATP in place of DNTPs. This will add one 3'A overhang. Purify DNA and
clone in a PCR cloning vector and slect clones for right insert. I
have faced such problem and solved in this way. all the best


On 1/20/10, methods-request from oat.bio.indiana.edu
<methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. Re: cross-linking antibodyies to beads (Irit Rappley)
>   2. seperating 3.5 and 3.3 kb in agarosegel (Senthil Thyagarajan)
>
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 19 Jan 2010 12:34:14 -0800
> From: Irit Rappley <irappley from scripps.edu>
> Subject: Re: cross-linking antibodyies to beads
> To: WS <novalidaddress from nurfuerspam.de>
> Cc: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
> Message-ID: <931CFBD1-5E9A-4045-AE11-5E5970F690F2 from scripps.edu>
> Content-Type: text/plain; charset="US-ASCII"; format=flowed
>
> Thanks! I will try your suggestions.
> Irit
>
>
> On Jan 18, 2010, at 11:02 PM, WS wrote:
>
> > Dear Irit,
> >
> > Seems to me your AB does not bind to protein G as desired, and AB
> > +target binds better.
> >
> > You might try covalent cross-linking instead of protein G. There are
> > e.g. carboxyl activated (magnetic) beads available. The coupling is
> > easy: buffer exchange your AB by gel filtration into e.g. borate
> > buffer, incubate with activated beads for 2hrs, quench and block, then
> > wash with your target buffer.
> >
> > Another option is biotinylating your AB and using streptavidin coated
> > beads. Procedure is similar as above.
> >
> > Concerning BSA as control, I'd prefer an unrelated antibody of the
> > same IgG subclass instead. Should have less possible "side effects".
> >
> > HTH
> >
> > Wo
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> ------------------------------
>
> Message: 2
> Date: Wed, 20 Jan 2010 11:02:08 +0100
> From: Senthil Thyagarajan <senthil.impres from googlemail.com>
> Subject: seperating 3.5 and 3.3 kb in agarosegel
> To: methods <methods from magpie.bio.indiana.edu>,   methods
>        <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <94d18fb41001200202r4d18f8fagfb3ad995048c7c70 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Dear all,
>
> I need to seperate DNA bands of size 3582bp and 3324bp and elute the former
> one. Can anyone suggest me the percentage of agarose gel, that could help me
> seperating these bands. Btw, there are no good restriction enzyme sites that
> break up the backbone and am left with no way other than seperating these
> two bands on the gel.
>
> Your help is very much appreciated. Please write to me asap. Thanks a lot in
> advance.
>
> Regards,
> Senthil
>
>
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> End of Methods Digest, Vol 56, Issue 15
> ***************************************
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-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210



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