Experiment 4:
Purification of DNA Fragment(s) by Electroelution after
Digestion and Separation on Agarose Gel
Theory:
Purification of DNA is required for cloning purpose. There are various methods
available for this purpose. However electroelution is quick and better in terms of
recovery. Electroelution means elution of DNA from agarose using electrical
current. The electroelution procedure allows one to purify very clean DNA to be
used in a large number of applications like sequencing, radio labeling, enzymatic
restriction, enzymatic modification, cloning etc.
Materials:
Electrophoretic chamber, power supply, dialysis membrane, TBE buffer, UV lamp,
vortex, microfuge, micropipettes (100 l, 20 l), micro tips, beakers, cylinder,
conical flask. 1-butanol, sodium acetate (2 M), ethanol (absolute, 70%), agarose,
glycogen, ethdium bromide (EB, 10 mg/ml, gel loading buffer; 0.25% bromophenol
blue, 0.25% xylene cyanol FF, 30% glycerol in distilled water, DNA markers. Clips
to tighten the dialysis bag.
Procedure:
1.
Cut out required band from agarose gel after separation and put into dialyzing
bag (previously boiled in distilled water.
2.
Close one end of the bag with clip, add 1-1.5 ml 1x TBE buffer, place the cut
band at one side of the bag and remove all the bubbles.
3.
Close the other end with clip, place the bag in the electrophoretic chamber and
run at least for 1-1 ½ at 80-100 V.
4.
Then check under UV lamp for complete DNA elution in the buffer (EB).
5.
Take out the buffer in a microcentrifuge and add equal amount of 1-butanol.
6.
Vortex thoroughly to mix two phases and centrifuge at room temperature for 3
minutes at 14,000 rpm.
7.
Remove aqueous phase (lower) with the help of pasture pipette/micro-pipette
into a clean microcentrifuge.
8.
Repeat the extraction (4-6 times) until pink color of EB disappears from both
aqueous and organic phase.
9.
Transfer the sample in microcentrifuge and add glycogen (10 g/10l), ½
volume 2 M sodium acetate and 2 volumes 100% ethanol.
10.
Leave on dry ice for 30 minutes or at –80o C overnight for precipitation.
11.
Centrifuge for 15 minutes at 4o C and wash with 100l 70% ethanol by spinning
for 5 minutes.
12.
Dry and reconstitute in 20 l autoclaved distilled water.
13.
Estimate concentration of purified DNA by running 5 l on agarose gel.
Discuss the Results:
http://www.jove.com/video/2136/electroeluting-dna-fragments