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DNA/RNA Extraction Protocol
Reagents
1.
TNE-Buffer 0.5% SDS
10%
Final
conc
DEPC
water 214.3 ml
4M NaCl
6.9 ml 0.11 M
1M Tris
pH 8.0 13.75 ml 55 mM
0.2M
EDTA pH8.0 1.375 ml 1.1 M
SDS
13.75 ml 0.55%
2.
Phenol H2O saturated
(Sigma)
3.
Chloroform: Iso-amyl Alcohol 50:1
4.
3M Na Acetate pH 5.2
5.
80% Ethanol
6.
Poly A 2mg/ml (carrier RNA)
7.
Proteinase K 10 mg/ml
Procedure
1.
Prepare extraction solution which
consists of TNE buffer with 0.5%SDS, 1mg/ml proteinase K, 40 ug/ml
PolyA. (Equivalent to 9 volumes of TNE: 1 volume Proteinase K: 20 ul
Poly A) For 12 tubes, this is 4.5 ml TNE, 500 ul Proteinase K, and
100 ul of Poly A
2.
Pre-incubate extraction solution for
10 minutes at 37oC to inactivate RNAses
3.
Add 100 ul of sample (serum/plasma)
and mix thoroughly
4.
Incubate for 10 minutes at 37oC
5.
Add 450 ul Phenol to each tube.
Place on a shaker for 5 minutes, and centrifuge at 13000 rpm for 5
minutes to separate the layers.
6.
Make up Chloroform/Iso-amyl alcohol
50:1 mix: For 10 tubes, this is 5 ml chloroform: 100 ul iso-amyl
alcohol.
7.
Transfer the aqueous (TOP) layer to
a fresh tube containing 450 ul chloroform/Iso-amyl alcohol. Place on
a shaker for 2 minutes. Centrifuge at 13000 rpm for 5 minutes
8.
Transfer the aqueous (TOP) layer to
a fresh tube containing 40 ul Na Acetate pH 5.2
9.
Add 800 ul (2 volumes) of ethanol
(-20oC) to each tube and mix thoroughly.
10.
Precipitate by incubating overnight
at -20oC or for 1 hour at -40o
11.
Collect nucleic acid extract by
centrifugating samples at 15000 rpm at 0oC for 10
minutes.
12.
Discard the supernatant. Wash pellet
with 600 ul 80% (v/v) Ethanol. Centrifuge again for 5 minutes at
15000 rpm to prevent loss of pellet.
13.
Dry the pellet at 42oC
(approximately 10-15 minutes) in the hot-block or PCR machine
14.
Re-dissolve the nucleic acid in 25
ul of nuclease free water and leave for 10 minutes before using in
any further reaction to ensure that the entire pellet is dissolved.
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