Mar 28, 2023

DNA - Ball Python DNA Extraction from sheds V.1

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzb344vx1/v1
DNA - Ball Python DNA Extraction from sheds
  • 1Lolita Genomics
Jose Avila Cervantes
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzb344vx1/v1
Protocol Citation: Jose Avila Cervantes 2023. DNA - Ball Python DNA Extraction from sheds. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb344vx1/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 15, 2022
Last Modified: March 28, 2023
Protocol  Integer ID: 71398
Keywords: ball python dna extraction, dna from ball python, ball python, dna, extraction, using phenol, python regius, isoamyl alcohol, dry shed
Abstract
Protocol to extract DNA from Ball Python (Python regius) dry sheds using Phenol:Chloroform:Isoamyl Alcohol
EQUIPMENT
  • Dry Bath / Heated Block
  • Microcentrifuge
  • DNA LoBind tubes 1.5mL
  • Micropipettes
  • Assorted pipette tips
REAGENTS

  • Lysis Buffer (10 millimolar (mM) Tris-base, 100 millimolar (mM) EDTA, 2% SDS, 5 Mass Percent , NaCl, 8 )
  • TE Buffer (EDTA 1 millimolar (mM) , Tris-Cl 10 millimolar (mM) )
  • Proteinase K (20 mg/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 900 µL of lysis buffer. Add 9 µL Proteinase K (20 mg/mL ) and vortex thoroughly for 00:00:30 seconds.

2. Incubate at 55 °C overnight .


30s
PHENOL/CHLOROFORM/ISOAMYL EXTRACTION
1. Add 500 µL of Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v) and vortex thoroughly for 00:00:30 seconds.

2. Centrifuge at room temperature for 00:05:00 at 16000 x g .

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

5m 30s
ETHANOL PRECIPITATION

1. Add 1000 µL of 100 % volume Ethanol, invert the tube and place the tube at -80 °C for 01:00:00 or at -20 °C Overnight .

2. Centrifuge the sample at 4 °C for 00:30:00 at 16000 x g to pellet the DNA.

3. Carefully remove the supernatant without disturbing the pellet.

4. Add 150 µL of 70 % volume ethanol.

5. Centrifuge the sample at 4 °C for 00:05:00 at 16000 x g .

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 00:05:00 to 00:10:00 .

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

1h 50m
DNA QUALITY CHECK

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

2. Add 4 µL Wide Range Ladder (100-12,000 bp) in the first well.
3. Premix 9 µL of sample and 1 µL of 10X loading dye or 5 µL of sample and 1 µL of 6X loading dye. Load this mixture in each well.

3. Run the samples for 00:40:00 at a 100V.

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

40m