Purifying small fragments - (Jan/23/2008 )

Hello all,

I am having trouble (gel) purifying small DNA fragments (<1 kb). We use a commercial kit, which works fine for eveyone here. However, when I do it my small fragments seem to dissappear. If at the same time I purify a larger fragment, it gives a fine yield, so it is not very probable that any of the materials is the culprit.

I have already tried using more elution buffer, using less elution buffer, different agarose concentrations in case of gel purification and heating elution buffer. We are working with AT-rich DNA and I do not use a cooled centrifuge, can this be a problem? Or are there any other things I might try?

TIA for your answers
David

Are you using a Qiagen kit? If so, try adding 1 volume of isopropanol to the QG-gel mix, this aids in recovery of both small and large fragments. If not, no idea, sorry!

Try a different kit for purification. Also try to elute in warm water or tris.

<1 kb is also big enough for gel purify........
which method are u using?

<1 kb is also big enough for gel purify........
which method are u using?

Did you add isopropanol?
Isopropanol is required when dealing with fragments smaller then 0.5kb and larger then 5kb

Qiagen kit require the addition of isopropanol to the agarose solvated in th orange QG buffer. The addition of isopropanol make a dramatic difference to the amount of DNA bound to the column.

In addition to the above, can you take the microfuge into the cold room?

Are you doing cloning? If so, restriction digest the DNA - separate bands in a low melting temperature gel in TAE buffer - cut out your target band under long wavelength UV quickly - melt the gel at 70-80*C - immediately put it in -80*C freezer for 15min - take out the tube and spin down the gel - take aliquot of the supernatant (that's your purified DNA fragment) to your ligase reaction. That's it - got your clone in 30min. No need to column purify your fragments. It's a waste of time and DNA.

I always use the 'squeeze method'.
Cut the band (from normal agarose, not low melting), put it into a syringe (whitout needle) and incubate o/n at -20 or a few h at -80. Remove the siringe and while the gel is softening squeeze it out in an eppendorf. Then phenol extract, chloroform extract and ethanol-Na acetate precipitate. If the fragments are very small use three volumes of ethanol.
It's longer, but it works really well.