DNA Recovery With Low Melt Agarose

Recovery of DNA from Low Melting Point Agarose Gels

1.Run digestion products on 0.7% LMP agarose gel in 1X TBE (it's nice to have at least 1ug of the fragment you want). LMP agarose is fragile; pour gel with EtBr

2.Let solidify in cold room. Be sure to overlay the gel with buffer before pulling out the comb, to prevent damage to the wells

3.Run gel in cold room, 100V

4.Cut out fragment of interest with clean razor blade and remove all excess agarose from the DNA.

5.Use long wave UV to visualize fragment when curring as this reduces nicking. Don't use UV box in the darkroom!

6.Melt gel slice at 65℃ 5min.

7.Determine volume, place back at 65℃.

8.Add 2.5 vol. of 20mM Tris pH8, 1mM EDTA, mix by pipetting, place at 65℃ 2min. [(10mL)(20mM)=x(1000mM) x=0.2mL; (10mL)(1?mM)=x500 x=20uL EDTA].

9.Place at RT for 5min.

10.Extract with equal vol phenol pH 7-8 (to prepare this, equilibrate 10ml phenol two times with an equal vol of 1M Tris pH8, followed by once with 100mM Tris pH8)

11.Extract DNA by shaking vigorously for 2min.

12.Spin 10min. at RT

13.Phenol/Chloroform extract (not pH7-8)

14.Chloroform extract

15.Precipitate with 1/10 vol 3M sodium acetate + 2vol Ethanol

16.Wash with 70% ethanol

17.Resuspend in 10ul water. Check recovery on minigel