Isolation of DNA fragments using glass milk (GENE-CLEAN)

1.Excise DNA section from gel into eppendorf tube.

2.Add 2-3 volumes of NaI (NaI for geneclean - dissolve 89.9 g NaI in 100ml dH2O store in light proof bottle) solution to gel fragment (best to be on the generous side) and place at 50 ℃ for 5' or until melted.Place glass milk on vortex or mixing wheel to resuspend.

3.Add 5 ul of glass milk to the dissolved gel slice - vortex and incubate for 5' at R.T.

4.Spin out glass milk 15 sec in microfuge and pour off the supernantant.

Optional For large gel fragments, rinse with 500ul NaI and leave 5' at 37℃, then spin and remove supernatant.

5.Add 0.5 ml of NEW WASH BUFFER, vortex and spin for 5 sec.and pour off the supernantant

6.Repeat step 5 twice.When the sample is vortexed the milk should evenly resuspend into a "fog".If you have not satisfactorily removed the agarose in step 4, then the glass milk will not resuspend properly after the second NEW wash rinse and you should start over by spinning out the glass milk and rinsing with NaI solution.

7.Pour off supernatant from the last rinse and respin.Remove the remainder of rinse with a Gilson but don't allow the pellet to dry.

8.Add 5 ul DDW, vortex and incubate for 5' at 37 ℃.Spin 5 seconds and collect TE containing DNA.

9.Repeat procedure once, without the 5' incubation.

10.Run aliquot on gel to see proportion recovered, which varies with the size of the fragment.Larger fragments (>4-5Kb) and very small (<0.3kb) give poor yields.NOTE.This procedure is probably not appropriate where a high proportion of fully intact, very large DNA is required eg.for microinjection, as fragments over 4-5 kb tend to be sheared (consider electroelution from gel slices instead).

Finebind (Amersham RPN 1621) modifications:

NaI solution:

90.8 g sodium iodide

1.5 g sodium sulphite

10 0ml distilled water (not made up 100 ml)

Filter sterile

Add a further 0.5 g sodium sulphite to ensure final solution is saturated.

Store in dark at 4 ℃ for no longer than 2 mths.

1.Melt agarose in NaI solution at 60℃

2.Add 10 ul finebind and allow finebind to bind DNA for 10' at RT.

3.Spin 15 sec and wash pellet with 0.5 ml NaI solution.

4.Wash three times with1ml 70% ethanol, 30% TE.

5.Elute with 5 ul DDW then 5 ul TE at 37℃ as above.