Screening for a 30bp difference using an agarose gel. - (Sep/21/2006 )

Hi all,

I first posted a few weeks ago about my unsuccessful cloning. When I attempted the construction again, I managed to obtain about two dozen colonies from the transformation mix (yay! I minimized UV exposure as much as possible and had a ratio of about 1:2). I did not get any colonies at all on the vector + ligase control but I used much less vector than in the vector + insert ligation mix because I was running low. Therefore, I am concerned that all my colonies are just going to give me back the source of the vector.

The complication lies in screening these colonies: the vector was made by cutting out 300bp from a pBAD24c plasmid. My insert is 270 bp. To screen, I cut the vector source and plasmids from my colonies so that I will obtain two fragments, one at 3190 bp with the other being either 1990bp or 2020 bp (depending on whether it has the new or old insert, respectively). I have tried running the digests on a 1% gel for an hour, comparing the positions of the bands from the vector source to bands from my colonies, then running another hour, checking every 15 minutes to see if there is any difference between the bands. The results were extremely inconclusive. The 3190 bp bands did not line up exactly so it was impossible to make any firm conclusions about the size of the other band.

Is there a way to screen for the new insert on a gel? If not, is there another method besides sequencing?

Thanks.

Hi,

couldn't you digest the 1990 or 2020 fragment further so you'll end up with something like 300 and 330? because the smaller the fragment the more easy the visualisation of the difference. further more with smaller fragments you can use PAGE which makes identification even easier

I can definitely cut it to make it smaller. I had never really thought about using PAGE because I am so used to associating it with running proteins.

Thank you very much

no problem...

dna PAGE gel should be 8-10% without SDS and as continious system. as running buffer I would recommend 0.5xTBE. You can run these at 20V/cm or so

I have used 3% agarose gel to seperate fragments, eg 330 and 375 bps.