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, [email protected] <[email protected]> wrote: > Send Methods mailing list submissions to > [email protected] > > 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 > [email protected] > > You can reach the person managing the list at > [email protected] > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: cross-linking antibodyies to beads (Irit Rappley) > 2. seperating 3.5 and 3.3 kb in agarosegel (Senthil Thyagarajan) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 19 Jan 2010 12:34:14 -0800 > From: Irit Rappley <[email protected]> > Subject: Re: cross-linking antibodyies to beads > To: WS <[email protected]> > Cc: "[email protected]" <[email protected]> > Message-ID: <[email protected]> > 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 > > _______________________________________________ > > Methods mailing list > > [email protected] > > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 2 > Date: Wed, 20 Jan 2010 11:02:08 +0100 > From: Senthil Thyagarajan <[email protected]> > Subject: seperating 3.5 and 3.3 kb in agarosegel > To: methods <[email protected]>, methods > <[email protected]> > Message-ID: > <[email protected]> > 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 > > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 56, Issue 15 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
