Help recovering 200bp DNA fragment

[Ron van Eijsden]

you at somehost.HSC.Colorado.edu (mike roper) wrote:
>In article <95216.110837VHWBC at CUNYVM.CUNY.EDU>, Harshinee Wijesinghe <VHWBC at CUNYVM.CUNY.EDU> says:
>>
>> 
>>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
>
>
>
><<snip>>
>
>Another (easy) method for recovering small fragments of DNA, in which we've been very succesfull, involves polyacrylamide electrophoresis:

1. Cut your DNA and load the digest on a TBE PAA-gel (which percentage of PAA
you should use for proper separation of your DNA is described in Maniatis).

2. Stain the gel for a short time with EtBr (after running)

3. Cut out the DNA fragment and elute overnight in an eppendorf tube in 200-500
ul 0.5M NH4Ac/1mM EDTA pH 8 at 55-60 degrees Celcius.

4. Next day, vortex for 1 minute and put the tube for 10 minutes in an
eppendorf centrifuge. Remove the PAA-slide and precipitate by adding 2 volumes
of ethanol.


Good luck!

Ron.