Help recovering 200bp DNA fragment

Harshinee Wijesinghe VHWBC at CUNYVM.CUNY.EDU
Fri Aug 4 10:08:37 EST 1995


 
I have been trying to elute and recover a 200bp insert (cut from a plasmid)
from agarose gel without much success. I definitely know that the DNA came
out of the gel as I monitored it under UV light and subsequently stained
the gel slice too. The protocol I used is as follows: After elution into
1.5 ml of TE buffer in dialysis bags I extracted with butanol and ether
before precipitating with ethanol. Lastly I used 0.01 MgCl2 with ethanol
at -70 degrees C overnight and spun for 30 min. After resuspension in 15 ul
of TE buffer and running a gel I find that although the insert is there the
band is very faint (i.e., not much has been recovered).
 
The problem does not seem to be with the elution but with the recovery
with ethanol. I would be very grateful for any suggestions for recovering
small (e.g. 200bp) fragments of DNA. Many thanks in advance.
 
 
Harshinee Wijesinghe
vhwbc at cunyvm.cuny.edu
 



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