RNA Extraction Protocol

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(Day 2)
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''Can also be used for DNA extraction by increasing phosphate buffer pH to ~8''
''Can also be used for DNA extraction by increasing phosphate buffer pH to ~8''
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''Change gloves often and use fume hood when working with phenol!''
+
''Change gloves often to prevent ribonuclease contamination''
 +
 
 +
''Use fume hood when working with phenol!''
 +
 
 +
''See bottom of page for modifications for environmental/Lake Washington sediment modifications''
 +
 
 +
 
 +
'''This protocol uses:'''
 +
*Ambion DNase I [https://products.invitrogen.com/ivgn/product/AM2222]
 +
 
 +
*Qiagen DNase I [http://www.qiagen.com/Products/Catalog/Lab-Essentials-and-Accessories/RNase-Free-DNase-Set#orderinginformation] prepared as directed and divided into tubes containing 50-70 ul each (prevents contamination and freeze/thaws of entire supply)
 +
 
 +
*Qiagen RNeasy Mini Kit [http://www.qiagen.com/Products/Catalog/Lab-Essentials-and-Accessories/RNeasy-Mini-Kit#orderinginformation]
==Day 1==
==Day 1==
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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 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)
 +
:''Prepare 2 tubes per batch-grown sample, 9-12 tubes per sediment sample''
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5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
5. Remove samples from centrifuge.  '''''Keep on ice!'''''   
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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  
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::(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
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::*If extracting RNA from sediment samples, add 5 ml to be able to resuspend soil pellet
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::Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes   
+
::*(''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)
7. Homogenise in a mini-beater (Biospec products) for 2 min  (3 min in less powerful beater)
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==Day 2==
==Day 2==
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12. Cool RNA room centrifuge
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13. Cool RNA room centrifuge
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13. Centrifuge frozen tube at 14,000 RPM (18,000 g) for 1 hour at 4°C
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Take Ambion DNase1 and buffer out of fridge and put on ice (starts thawing)
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14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for '''1 hour''' at 4°C
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14. Remove supernatant and add 500 μl of 75% ethanol
+
:Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap) and buffer out of freezer and put on ice to thaw
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15. Centrifuge at 18,000 g for 5 min at 4°C
+
 
-
16. Remove supernatant and centrifuge at 18,000 g for 2 min at 4°C
+
15. Remove supernatant and '''add 500 μl of 75% ethanol'''
-
17. Remove any liquid residue from the tubes by pipetting  
+
 
-
Dry samples for 15 min at RT with caps off (evaporating alcohol)
+
16. Centrifuge at 14,000 rpm for 5 min at 4°C
-
18. Pool every 2 samples (2-3 for LWS samples, 1 otherwise):  
+
 
-
Re-suspended in 100μl of DNase I mix  
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17. Remove supernatant and centrifuge at 14,000 rpm for 2 min at 4°C
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(Make one master tube containing: 90 μl ddH2O, 10 μl of 10xDNase buffer and 5 μl DNaseI per pool)
+
 
-
19. Incubate at 37oC for 30 min
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18. Remove any liquid residue from the tubes by pipetting  
-
20. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)  
+
 
-
21. For LWS pooled samples, do the following twice:
+
19. Dry samples for '''15 min''' at RT with caps open (evaporating alcohol)
 +
 
 +
20. Pool every 2 samples (2-3 for sediment samples, 1 otherwise):  
 +
:Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
 +
::Ambion DNase master mix:
 +
::*90 ul ddH2O
 +
::*10 ul Ambion 10xDNase buffer
 +
::*5 ul DNase I enzyme
 +
 
 +
21. Incubate at 37°C for '''30 min'''
 +
 
 +
22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)
 +
 
 +
23. For LWS pooled samples, do the following twice:

Revision as of 18:37, 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 to prevent ribonuclease contamination

Use fume hood when working with phenol!

See bottom of page for modifications for environmental/Lake Washington sediment modifications


This protocol uses:

  • Ambion DNase I [1]
  • Qiagen DNase I [2] prepared as directed and divided into tubes containing 50-70 ul each (prevents contamination and freeze/thaws of entire supply)
  • Qiagen RNeasy Mini Kit [3]

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 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)
Prepare 2 tubes per batch-grown sample, 9-12 tubes per sediment sample

5. Remove samples from centrifuge. Keep on ice!

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

  • If extracting RNA from sediment samples, add 5 ml to be able to resuspend soil 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 to allow full precipitation of nucleic acids.

Day 2

13. Cool RNA room centrifuge

14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for 1 hour at 4°C

Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap) and buffer out of freezer and put on ice to thaw

15. Remove supernatant and add 500 μl of 75% ethanol

16. Centrifuge at 14,000 rpm for 5 min at 4°C

17. Remove supernatant and centrifuge at 14,000 rpm for 2 min at 4°C

18. Remove any liquid residue from the tubes by pipetting

19. Dry samples for 15 min at RT with caps open (evaporating alcohol)

20. Pool every 2 samples (2-3 for sediment samples, 1 otherwise):

Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
Ambion DNase master mix:
  • 90 ul ddH2O
  • 10 ul Ambion 10xDNase buffer
  • 5 ul DNase I enzyme

21. Incubate at 37°C for 30 min

22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)

23. For LWS pooled samples, do the following twice:

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