How to determine appropriate agarose % - (Jun/08/2005 )
I wonder if there's any standard comparison table/graph available for the determination of (agarose %) used to achieve best resolution of desired fragment sizes. For example, my past advisor had told me to use the following setups for my experiment, but he also said this is only roughly correct and everyone should have his own comparison table to follow, thanks.
(fragment size) (% agarose used)
>1.5 kb 0.8%
>0.5 kb, but <1.5 kb 1.0%
<0.5 kb 1.2%
I'm now encountering some problem as what I should do (w/o the use of PAGE 'cause my current lab doesn't have this sort of equipment) to separate two DAN fragments of similar sizes (120 and 80 bp). Someone had suggested that I use 4% gel to get them resolved, however, I found gel of this high concentratio was very difficult using either microwave or hot plate to prepare. I've tackling on this issue for weeks and any suggestion would be gratefully appreciated. Thanks.
i would try a 3.5% and a long separation distance...
What we sometimes do is on 4% percent gels we will make up with 3% standard agarose and 1% low melting temperature agarose. ie. In 100ml use 3g of standard agarose and 1g of low melting point agarose.
Hope this helps
check out Ambion website tech note, they have a sheet called something like 'Which Agarose to use' it gives a list of %'s for PA and agarose. Thats what i go buy, for short sequences you would use a sieving agarose of 3-4%, Invitrogen have a 10bp marker and they separate it on a 3-4% gel, check out their site, the marker may also be helpful.
Why not try a different solution: Do you need both the 120bp AND 80bp bands or just one? If you just need, say, the 120bp band, find a restriction enzyme that cuts the 80bp band near the middle, but does not cut in the 120bp band. Then you can just purify the one band. I've done this before to get rid of vector bands that were near the size of the inserts I was trying to purify. Hope this helps.
Want to be clever?
Run them out on a 5% non denaturing acrylamide gel. You can still extract both fragments with the 'crush and soak' method.
You will get wonderful separation. Trust me.
I used 4% agarose-1000 (low melting temperature agarose; Invitrogen) electrophoresis for separate DNA fragments from restriction enzymatic digestion(Hae III, Bst EII and Msp I).Because of size of bands vary from 60 – 330 bp.
Metaphor or Nusieve 3:1 agarose can be used to easily separate fragments of this size.
phage434 is absolutly correct.
I would do this before my clever little acrylamide trick.