PCI Extraction

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