Reagents and Solutions
TESC Buffer | 11 mL/sample |
Proteinase K | 30 µL/sample |
10% SDS | 550 µL/sample |
PCI | ~15 mL/sample |
CI | ~10 mL/sample |
Isopropanol | ~6 mL/sample |
70% Ethanol (-20oC) | ~0.5 mL/sample |
10 mM Tris or TE Buffer | 250 µL/sample |
Materials
Scalpel, autoclaved | 1 |
Tweezers, autoclaved | 1 |
Weigh Boats, autoclaved | 1 per sample |
All-Glass Tissue Grinder or ceramic mortar and pestle, autoclaved | 1 per sample |
1.5 mL Eppendorf Tubes, autoclaved | 1 per sample |
100 µL Pipette | 1 |
100 µL Pipette Tips, autoclaved | one per sample |
1 mL Pipette | 1 |
1 mL Pipette Tips, autoclaved | many |
5 mL PCI-Pipette (located in the hood in LSE404) | 1 |
5 mL Pipette Tips, autoclaved | many |
Water Baths | one a 37oC one at 50oC one at 65oC |
50 mL Plastic Centrifuge Tubes with Screw Caps | four per sample |
Sample Preparation
- Autoclave everything
- Using sterile tweezers, scalpel and weigh boat, weigh out approximately 50-300 mg wet mass of sample (flame sterilize tweezers and scalpel between samples)
- Transfer sample into all-glass tissue grinder (potter), add 1 mL TESC buffer and homogenize
- Using 1 mL pipette, transfer homogenate to 50 mL centrifuge tube
- Rinse potter with 4 mL TESC buffer and combine with above homogenate
- Rinse potter with 1 mL TESC buffer and combine with above homogenate
- Freeze at -80oC (~20 minutes), thaw at room temperature for ~5 minutes, thaw completely in 65oC water bath (~10 minutes)
- Repeat above sequence five times (for a total of six freeze-thaws)
Extraction
- Add 30 µL Proteinase K solution and 550 µL 10% SDS solution to sample
- Gently mix by inverting capped tube several times
- Incubate at 50oC for 40 minutes, gently mixing every 10 minutes
- Add 5 mL PCGently mix by inverting capped tube several times
- Centrifuge at 4,100 rpm for 10 minutes
- Carefully transfer aqueous (ie top) layer into a new 50 mL centrifuge tube
- Add 5 mL TESC buffer to original tube
- Gently mix by inverting capped tube several times
- Centrifuge at 4,100 rpm for 10 mintues
- Combine aqueous (ie top) layer with that from above
- Add one volume (~10mL) PCI to combined aqueous layers
- Gently mix by inverting capped tube several times
- Incubate at 65oC for 10 minutes, gently mixing every five minutes
- Centrifuge at 4,100 rpm for 12 minutes
- Transfer aqueous (ie top) layer into a new 50 mL centrifuge tube
- Add one volume (~10mL) CI
- Gently mix by inverting capped tube several times
- Centrifuge at 4,100 rpm for 12 minutes
- Transfer aqueous layer to new 50 mL centrifuge tube
- Centrifuge at 4,100 rpm for 12 minutes
- Remove any remaining PCI/CI from tube
- Gently mix by inverting capped tube several times
- Centrifuge at 4,000 rpm for 8 minutes
Culmination
- Precipitate nucleic acids with 0.6 volume (~6 mL) isopropanol for at least 30 minutes at room temperature, or overnight at 4oC
- Alternatively, add 0.04 volume (~400 µL) 5 M NaCl and mix, add 2 volumes (~20 mL) cold (-20oC) 100% ethanol and mix, precipitate for 60 minutes in an ice bath
- Centrifuge at 4,100 rpm for 60 minutes at 4oC
- Decant supernatant
- Wash pellet with cold (-20oC) 70% ethanol, pouring it slowly along the tube’s wall. If pellet dislodges, re-centrifuge at 4,100 rpm for 20 minutes at 4oC
- Dry pellet for 1-3 hours in vacuum
- Add 250µL 10 mM Tris (or TE) buffer to tube
- Incubate at 37oC for at least 30 minutes to allow for dissolution
- Transfer re-suspended pellet into a 1.5 mL Eppendorf tube
- Store at -20oC
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