Extracting DNA from a gel

1.Cut out a DNA fragment from the agarose gel and place the gel piece into a microfuge tube

2.Add 500-1000ul NaI buffer (at least 3x the gel piece volume)

3.Incubate at 55ºC for ~10 min with occasional shaking, until the gel piece dissolves in the buffer

4.Add 1-3ul glass milk and mix the sample by either vortexing or vigorous shaking

5.Incubate on ice for ~10min with occasional shaking to allow the DNA to bind to glass beads

6.Centrifuge for 20 sec @ top speed to pellet glass beads with bound DNA

7.Aspirate supernatant taking care to suck dry all of the supernatant drops from the walls

8.Add 200ul of cold NaI buffer, shake in Vivadent shaker for 2 sec, and centrifuge for 20 sec to pellet glass:DNA

9.Aspirate away supernatant, add 700ul of cold NEET wash, shake in Vivadent shaker for 2 sec, centrifuge for 20 sec to pellet glass:DNA, apsirate the wash

10. Repeat step 9 two more times (for the total of three NEET washes), keeping samples on ice

11.Spin the samples again for 1 min and aspirate all of the remaining NEET wash

12. Air dry samples for ~15 minutes by leaving microfuge tubes caps open at RT

13. Elute the DNA samples by adding 10-20ul of di water: resuspend the pellets by shaking in the Vivadent shaker for 2 sec, knock on the tubes to bring the drops to the bottom of the tubes, and incubate the samples at 55ºC for 5-10 min

14. Centrifuge the samples for 1-2 min to collect glass beads on the bottom of the tube

15. Carefully transfer the supernatant containing eluted DNA to a fresh microfuge tube taking care not to transfer glass slurry.

16. Check DNA recovery and estimate DNA concentration by running 1ul of the sample on agarose gel.

NaI buffer (100ml):

90.8 g NaI

1.5 g Na2SO4

Add hot di water up to 100ml

Stir in the dark (the solution is light sensitive)

Filter through Whatman paper to remove remaining crystals of salt (the solution is saturated, so not all of the salt will dissolve)

Store in a dark bottle at 4ºC

NEET wash (500 ml):

10ml 5M NaCl

1ml 0.5M EDTA

250ml 95% EtOH

5ml 1M Tris-HCl pH 7.5

234 ml di water

Mix, and store at –20ºC