DNA?Fragment?Purification

Materials:

1.TE solution

o10 mM Tris (pH to 7.5)

o1 mM EDTA (pH to 8.0 to dissolve)

2.Frozen agarose gel piece containing the desired DNA fragment

Supplies:

1.Micropipetter and tips

2.Microcentrifuge and tubes

3.Spatula

4.PCR machine and the 600-ul tubes for use in the machine

5.Ultrafree-MC(R)filter units,0.45μm

Procedures:

1.Quick thaw the gel piece in a 600-ul tube using the PCR machine at 37 degrees C

2.Macerate the gel piece with a spatula.

3.Transfer into the sample cup of the filter unit.

4.Spin the filter unit for 20 minutes at 5,000 g.

5.Transfer fluid in the bottom of the filter unit to a new tube and place on ice.

6.Add 200μl of TE to the sample cup of the filter unit and incubate at room temperature for 10 minutes.

7.Spin the filter unit for 20 minutes at 10,000 g.

8.Combine with the first collection and estimate the total volume.

9.Add 0.1 volume of the sodium acetate and 3 volumes of 100 % ethanol.

10.Store the tubes at -20 degrees C for 1 hour.

11.Spin in a microcentrifuge at maximum speed for 15 minutes.

12.Discard supernatant and add 400μl of 70 % ethanol.

13.Spin in a microcentrifuge at maximum speed for 5 minutes.

14.Discard the ethanol and add another 400μl of 70 % ethanol.

15.Spin in a microcentrifuge at maximum speed for 5 minutes.

16.Withdraw and discard as much liquid (ethanol)as possible then air-dry the pellet.

17.Resuspend the pellet in 20μl of distilled water and keep at 4 degrees C or -20 degrees C for storage and for estimation of DNA concentration (LAB 4)later.

Results:

1.This method of DNA fragment purification works well with fragment sizes less than 5,000 basepairs.The yield,based on subsequent estimation of DNA concentration (LAB 4),is typically 50% or better depending on the size.