how to gel-purify a 48kb fragment - (Jan/24/2006 )
In a cloning experiment I have to clone a 48kb fragment.
How can I purify this fragment from an agarose-gel since extensive vortexing will break my fragment?
Suggestions?
For qiagen gel purification I don't vortex actually. I know that it doesn't allow more than 10 kb, but at least I can do it without the vortexing. Just make sure that your agarose is nicely dissolved in the buffer before continuing (I assume you have a kit/extraction method available that allows to purify DNA this size).
How can I purify this fragment from an agarose-gel since extensive vortexing will break my fragment?
Suggestions?
Have you thought of using AGARASE?
If not then this is the time to switch to Agarase, painful part is you will have to use LMP.
I know of three alternatives, all using electro elution
1. Electro-Elution with Elutrap from Schuell & Schleicher - 1500 Euro for the thing, tested with 50Kb fragment by someone here
2. Gebaflex electro elution tubes from Gerard Biotech ( http://www.gerardbiotech.com/electoelution.htm ) not tested yet by myself but thinking about getting some maybe, allows to perform parallels without another Elutrap after all ...
3. Make a 1% TAE gel, let it run, cut the band out. Make a second 1% gel, cut a pocket of the right size and around 4 cm long, place your band inside and let it run 30" 200V, you can run a test elution with a mobile UV bulb to get a feel for it. Of course 1x TAE running buffer but only till the upper end of the gel and pipetting TAE into the pocket too. I tried that but haven't got a precipitation so far, no idea what the problem is, tweaking this, protocoll is from a friend.