electrophoresis to resolve two DNA of similar size - (Mar/30/2006 )
hello, i need to resolve two different sized bands using electrophoresis; 2150 and 2200 bp. for reasons too complicated to explain here, i cant use restriction enzymes or anything like that.
any ideas are welcome. ive tried agarose with tae ans sb and also poliacrylamide and nothing!
please help, this is all i need to finish my thesis
thax.
any ideas are welcome. ive tried agarose with tae ans sb and also poliacrylamide and nothing!
please help, this is all i need to finish my thesis
thax.
Hello!
I am not sure if this is going to work ut is what i whould do in your position.
Try an agarose gel of a quiet high percentage (eg. 2%) and use a big frame of 20cm or more (that is a big one and not the small normally used). Then you let your gel run for quiet a long time at 90V , it is going to take some time because your bands are quite big, but it would permit the separation!
hello, i need to resolve two different sized bands using electrophoresis; 2150 and 2200 bp. for reasons too complicated to explain here, i cant use restriction enzymes or anything like that.
any ideas are welcome. ive tried agarose with tae ans sb and also poliacrylamide and nothing!
please help, this is all i need to finish my thesis
thax.
Hello!
I am not sure if this is going to work ut is what i whould do in your position.
Try an agarose gel of a quiet high percentage (eg. 2%) and use a big frame of 20cm or more (that is a big one and not the small normally used). Then you let your gel run for quiet a long time at 90V , it is going to take some time because your bands are quite big, but it would permit the separation!
Do not put to much DNA because the size of the band is very similar and fusion beteen them is very probable.
mmm... tried that... thats why im trying with polyacrilamide now 
Yes, better to use a very small amount of DNA, to get very thin bands, and a larger amount of EtBr (up to ~150 ug EtBr/ 50 ml gel, or equivalent amount for staining after you run the gel) or SYBR Green I and/or long exposures to see them. (Probably you've already thought of this, but I thought I'd throw it out there.)
Do you have to use electrophoresis? Could you use Prep LC-MS? That way you could isolate the fragment by molecular mass and get a pretty clean separation.
You can try DGGE- Denaturing gradient gel electrophoresis- or SSCP - Single stranded conformation polymorphism assays.
They are polyacrylamide type gels, with acrylamide(SSCP) and urea concentration(DGGE) gradients in them. It is a dated technique, but you can certainly discriminate fragments with very little size difference.