Electrophoresis, pls urgent - (Jun/13/2007 )

Hello all, I would like to resolve out 2 DNA fragments, one is 9.23kb and other is 8.62kb..pls what would be the best condition for gel and electrophoresis.......thank you..

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i think its very very difficult if you use agarose gel

once i seperate 4.4 kb from 4.9 and it was tough
i used .5% long agarose gel and run it at 50 volt for 3 hours

i dont know if you can use native page to seperate this, you can enquiry about it

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Can try a 2% agarose (1% regular agarose + 1% metaphor). Run in the longest tray that you have. The time will depend of the lenght of the tray and the V should be less than 95 (the tray is very large), 75-85 for a med size and 50 for the tiny one. run until you don't see the dye.

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Hello, thank you very much for your useful information.....I never heard about mixing the regular agarose and metaphor.....

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heyy its very easy if it is plasmid..

Digest one plasmid with an unique restriction site.. u'll see two bands..

what DNA it is?

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this is a plasmid but...bit complicated. I am doing partial digestion of a 9kb leniarized(NcoI) plasmid with BamHI since it got 2 Bam HI sites. I want to digest out a 800bp fragment (NcoI-BamHI) and get the backbone out to clone another fragment to the same site...when I am digesting the leniarized fragment, I am mainly getting single cutband (9kb) 5kb, 4kb and 0.8kb bands but not the regured one to elute 8.2 kb. this is all the story....so please any of you got any comments please help me with it..thank you

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this will be tough. try a 0.5% and run through out the day at 20 V or overnight in a large chamber. you may succeed.

good Luck !!!

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heyy i got an idea... lol

transform your DNA mixture in bugs.. n then do the colony PCR to recognise ...which bug is having what DNA..

grow the bug and do the maxi.. enjoy..



but a simple problem here.. if you got both DNA in same bug..

no worries..

test the same bug with both plasmid primers.. and select the colony which is single positive only..



what do you think??? i think its possible and very easy..

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The plan is possible but I don't think it will be easy. The linearised NcoI (9.23kb) vector will very likely religate more readily, then the partial digest vector (BamHI/NcoI) will ligate to the new insert.

So unless the linearised NcoI vector can be removed or decreased in some way, the number of vector+new insert colonies will be vastly outnumbered by the religated vector. Colony Southernblot will definately resolve this or a large colony PCR (you will definately need multichannel pipettes to do this). I would screen ~200+ colonies

However, if the new insert can kill the NcoI site. An NcoI digest after ligation will fix this problem. FatI. PciI digest? Nope?

If the partial digest bands can not be resolved, and the strategy suggested by LaboratoryHD doesn't work, you could monkey around with the vector.

Assuming that your vector does not have a BglII site.
You could ligate the NcoI cut vector with an oligo which carries a BglII site. Importantly one of the primers does not reform the NcoI site. It kills the site. While the second primer keeps the site.

Orientation of the insert has to be done by PCR. One of the primers used to make the site and one primer binding to the vector.

Finally,another alternative would be to avoid the problem and go hunt for an empty vector.

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I alrady thought about this, but lot of colony screening....the idea which suggested by perneseblue looks much easier...any way thanks for sharing your idea.....

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