©Michael Court
Created Dec 2002
PCR product purification by DNA Precipitation
with Linear Acrylamide
1. Use this technique for purifying short (<200 bp) PCR product prior to sequencing
or other use that requires removal of dNTPs and primers.
a. This is instead of Qiagen spin columns since recoveries with spin columns
can be very poor with short DNA fragments
b. Linear acrylamide is used as a co-precipitant to enable ready visualization
of the pellet and enhance recovery
c. Linear acrylamide should not interfere with “downstream” reaction.
2. In 1.5 mL microcentrifuge tube
a. Add 100 uL PCR product
i. Other volumes can be scaled proportional to PCR product volume
b. Add 10 uL 3 M sodium acetate
c. Vortex
d. Add 4 uL of 0.5 ug/uL linear acrylamide (final conc 10-20 ug/mL;
Ambion)
e. Vortex
f. Add 228 uL 100% ethanol (2 volumes)
g. Vortex
3. Incubate on dry ice for 15 minutes
4. Centrifuge at > 10,000 g for 5 minutes
5. Discard supernatant
a. Watch out for tiny white pellet
6. Wash pellet with 500 uL of 80% ethanol
7. Centrifuge at > 10,000 g for 5 minutes
8. Discard as much of supernatant as possible
9. Dry pellet
a. Leave on bench with top open for 1-2 hrs
b. OR place in vacuum oven for 15 min
10. Add 20 – 30 uL of water or TE buffer
11. Store in freezer or refrigerator until use.