Difference between revisions of "RNA Extraction Protocol"

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(Day 1)
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==Day 1==
 
==Day 1==
# Start centrifuge cooling with 50 mL tube holders
+
1. Start centrifuge cooling with 50 mL tube holders
# Add 5 mL of cold stop solution to a fresh 50 mL tube  
+
 
 +
2. Add 5 ml of cold stop solution to a fresh 50 mL tube  
 
::(''stop solution'' = 5% buffer equilibrated phenol [pH 7.4] in ethanol)
 
::(''stop solution'' = 5% buffer equilibrated phenol [pH 7.4] in ethanol)
3. Add sample up to 50 mL
+
 
 +
3. Add sample up to 50 ml
 +
 
 
4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C
 
4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C
::While centrifuging, prepare 2 ml screw-cap tubes (2 tubes per sample) with:
+
:'''While centrifuging''', prepare 2 ml screw-cap tubes (2 tubes per sample) with:
 
:::*0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
 
:::*0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
 
:::*35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
 
:::*35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
 
:::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
 
:::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
 
5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
 
5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
 +
 
6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet   
 
6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet   
 
::(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
 
::(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
::Mix well and transfer 1mL aliquots into prepared 2 ml screw-cap tubes   
+
::Mix well and transfer 1 mL aliquots into prepared 2 ml screw-cap tubes   
 +
 
 
7. Homogenise in a mini-beater (Biospec products) for 2 min  (3 min in less powerful beater)
 
7. Homogenise in a mini-beater (Biospec products) for 2 min  (3 min in less powerful beater)
 +
 
8. Centrifuge at 14000 rpm for 5 min at 4°C  
 
8. Centrifuge at 14000 rpm for 5 min at 4°C  
 +
 
9. Transfer the upper aqueous phase into a new 2 mL tube   
 
9. Transfer the upper aqueous phase into a new 2 mL tube   
 
::Add 750µL chloroform:isoamylic alcohol (24:1)
 
::Add 750µL chloroform:isoamylic alcohol (24:1)
 +
 
10. Centrifuge at 14000rpm for 5 min at 4oC.
 
10. Centrifuge at 14000rpm for 5 min at 4oC.

Revision as of 11:32, 22 May 2013

Extraction Protocol modified from Griffiths et. al, 2000

Can also be used for DNA extraction by increasing phosphate buffer pH to ~8

Change gloves often and use fume hood when working with phenol!

Day 1

1. Start centrifuge cooling with 50 mL tube holders

2. Add 5 ml of cold stop solution to a fresh 50 mL tube

(stop solution = 5% buffer equilibrated phenol [pH 7.4] in ethanol)

3. Add sample up to 50 ml

4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C

While centrifuging, prepare 2 ml screw-cap tubes (2 tubes per sample) with:
  • 0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
  • 35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
  • 750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)

5. Remove samples from centrifuge. Keep on ice!

6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet

(extraction buffer = [10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
Mix well and transfer 1 mL aliquots into prepared 2 ml screw-cap tubes

7. Homogenise in a mini-beater (Biospec products) for 2 min (3 min in less powerful beater)

8. Centrifuge at 14000 rpm for 5 min at 4°C

9. Transfer the upper aqueous phase into a new 2 mL tube

Add 750µL chloroform:isoamylic alcohol (24:1)

10. Centrifuge at 14000rpm for 5 min at 4oC.