Mar 27, 2023

Public workspaceDNA - Ball Python DNA Extraction from sheds V.1

DOI
dx.doi.org/10.17504/protocols.io.rm7vzb344vx1/v1
  • 1Lolita Genomics
Jose Avila Cervantes
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DOI: 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 27, 2023
Protocol Integer ID: 71398
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 (Concentration10 millimolar (mM) Tris-base, Concentration100 millimolar (mM) EDTA, 2% SDS, Concentration5 Molarity (M) , NaCl, Ph8 )
  • TE Buffer (EDTA Concentration1 millimolar (mM) , Tris-Cl Concentration10 millimolar (mM) )
  • Proteinase K (Concentration20 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 Amount900 µL of lysis buffer. Add Amount9 µL Proteinase K (Concentration20 mg/mL ) and vortex thoroughly for Duration00:00:30 seconds.

2. Incubate at Temperature55 °C overnight .


30s
Incubation
Digestion
Overnight
PHENOL/CHLOROFORM/ISOAMYL EXTRACTION
1. Add Amount500 µL of Phenol/Chloroform/Isoamyl Alcohol 25:24:1 (v/v) and vortex thoroughly for Duration00:00:30 seconds.

2. Centrifuge at room temperature for Duration00:05:00 at Centrifigation16000 x g .

3. Carefully remove the upper aqueous phase (~Amount500 µL )and transfer the layer to a fresh tube. Be sure not to carry over any Phenol during pipetting.

5m 30s
Toxic
ETHANOL PRECIPITATION

1. Add Amount1000 µL of Concentration100 % volume Ethanol, invert the tube and place the tube at Temperature-80 °C for Duration01:00:00 or at Temperature-20 °C DurationOvernight .

2. Centrifuge the sample at Temperature4 °C for Duration00:30:00 at Centrifigation16000 x g to pellet the DNA.

3. Carefully remove the supernatant without disturbing the pellet.

4. Add Amount150 µL of Concentration70 % volume ethanol.

5. Centrifuge the sample at Temperature4 °C for Duration00:05:00 at Centrifigation16000 x g .

6. Carefully remove the supernatant without disturbing the pellet.

7. Repeat Concentration70 % volume ethanol wash once and remove as much of the remaining ethanol as possible.

8. Dry the DNA at TemperatureRoom temperature for Duration00:05:00 to Duration00:10:00 .

9. Re-suspend the DNA pellet in Amount200 µL of TE Buffer.

1h 50m
Wash
DNA QUALITY CHECK

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

2. Add Amount4 µL Wide Range Ladder (100-12,000 bp) in the first well.
3. Premix Amount9 µL of sample and Amount1 µL of 10X loading dye or Amount5 µL of sample and Amount1 µL of 6X loading dye. Load this mixture in each well.

3. Run the samples for Duration00:40:00 at a 100V.

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

40m
Analyze