problems with purifying a 100 bp DNA fragment - (Dec/18/2006 )

Hy everybody

I am having problems with a purification of a 100 bp PCR product.
I tried different commercial kits (PCR purification or Gel purification) and my fragment pass thrhough the column!!, so I don't get nothing at all in the elution step...

Have you ever experienced something like that? What can I do to get my digested product pure? I need to ligate it into a vector then, but I have to get rid of restrictions enzymes before...

Any help would be greatly appreciated...

Have you tried beads? You can also get columns specifically for small fragments. Look up the major companies for purification of small DNA fragments. Or melt the agar with your yellow liquid, then precipitate the old fashioned way.
Fiona.

Would follow fgoulds advise concerning precipitation.

Try and see if a centrifuge ultrafiltration unit with MWCO 30,000 would help.

Depending of the kit you're using, it is logical that you loose such a small fragment... For instance if I remember well, the cut off for the qiagen PCR cleanup kit is 200 bp...
If your PCR fragment is clean enough (if you don't have any non-specific band), you can try to heat inactivate your restriction enzymes and do nothing more...

You can try the freeze/squeeze.
http://www.bios.niu.edu/core/freeze_squeeze.htm

Alternatively, just cut out the gel band, place it in a aerosol barrier tip 200ul size (cut off the tip). Place in eppendorf and centrifuge. The liquid that comes through contains your DNA and can be used directly for ligation.

Recently I had amplified 100bp fragment.

I did the PCR, ran on gel and purified it and then digested it and again ran on gel and purified it and used it for ligation. Worked nicely.

I used Invitrogen gel purification kit, if this can b of any help.

Ammonium acetate is good for small fragments. Add 1/2 volume 7.5 M ammonium acetate, pH 7.5 and 2 vol 95% EtOH.

Qiaquick DNA cleanup kit from Qiagen works for me. Their recovery range is from 100bp - 10kb.
I have routinely purified 100bp fragment their qiaquick column.
Checked on gel before and after purification and recovery is also good.
Hope it helps.