DNA - Ball Python DNA Extraction from sheds

EQUIPMENT

• Dry Bath / Heated Block
• Microcentrifuge
• DNA LoBind tubes 1.5mL
• Micropipettes
• Assorted pipette tips

REAGENTS

• Lysis Buffer (10millimolar (mM) Tris-base, 100millimolar (mM) EDTA, 2% SDS, 5Molarity (M), NaCl, 8 )
• TE Buffer (EDTA 1millimolar (mM), Tris-Cl 10millimolar (mM) )
• Proteinase K (20mg/mL )
• Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v)
• Ethanol 100 %
• Ethanol 70 % (Freshly prepared)
• Tris Acetate-EDTA (TAE) Buffer
• SYBR Safe DNA stain
• Agarose
• Loading Dye
• Wide Range Ladder (100-12,000 bp)

PROTEINASE K DIGESTION

1. Cut a small piece of fresh tissue (3-5mm). Put in a 1,5mL microtube. Add 900µL of lysis buffer. Add 9µL Proteinase K (20mg/mL ) and vortex thoroughly for 0h 0m 30s seconds.

2. Incubate at 55°C .

PHENOL/CHLOROFORM/ISOAMYL EXTRACTION

1. Add 500µL of Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v) and vortex thoroughly for 0h 0m 30s seconds.

2. Centrifuge at room temperature for 0h 5m 0s at 16000x g,0h 0m 0s .

3. Carefully remove the upper aqueous phase (~500µL )and transfer the layer to a fresh tube. Be sure not to carry over any Phenol during pipetting.

ETHANOL PRECIPITATION

1. Add 1000µL of 100% volume Ethanol, invert the tube and place the tube at -80°C for 1h 0m 0s or at -20°C .

2. Centrifuge the sample at 4°C for 0h 30m 0s at 16000x g,0h 0m 0s to pellet the DNA.

3. Carefully remove the supernatant without disturbing the pellet.

4. Add 150µL of 70% volume ethanol.

5. Centrifuge the sample at 4°C for 0h 5m 0s at 16000x g,0h 0m 0s .

6. Carefully remove the supernatant without disturbing the pellet.

7. Repeat 70% volume ethanol wash once and remove as much of the remaining ethanol as possible.

8. Dry the DNA at Room temperature for 0h 5m 0s to 0h 10m 0s .

9. Re-suspend the DNA pellet in 200µL of TE Buffer.

DNA QUALITY CHECK

1. Prepare a 1% agarose gel with 1X TAE buffer and 1X SYBR Safe DNA stain.

2. Add 4µL Wide Range Ladder (100-12,000 bp) in the first well.

3. Premix 9µL of sample and 1µL of 10X loading dye or 5µL of sample and 1µL of 6X loading dye. Load this mixture in each well.

4. Run the samples for 0h 40m 0s at a 100V.

5. Visualize in a trans illuminator and take a photo of the gel.