Reagents and Solutions

Materials

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