RNA Extraction Protocol

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Day 1)
(Day 1)
Line 22: Line 22:
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 1 mL 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)
Line 28: Line 28:
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 750 µl chloroform:isoamylic alcohol (24:1)
-
10. Centrifuge at 14000rpm for 5 min at 4oC.
+
10. Centrifuge at 14000 rpm for 5 min at 4°C.
 +
:'''While centrifuging''', prepare tubes for precipitation (these will go in -80°C freezer)
 +
::*5 ul 0.5M MgCl2
 +
::*75 ul 3M sodium acetate
 +
11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol
 +
 
 +
12. Incubate overnight at -80°C.

Revision as of 16:26, 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 750 µl chloroform:isoamylic alcohol (24:1)

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

While centrifuging, prepare tubes for precipitation (these will go in -80°C freezer)
  • 5 ul 0.5M MgCl2
  • 75 ul 3M sodium acetate

11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol

12. Incubate overnight at -80°C.

Personal tools