Mar 27, 2023

Public workspaceDNA - Ball Python DNA Amplification with TFEC primers V.1

DOI
dx.doi.org/10.17504/protocols.io.3byl4jb78lo5/v1
  • 1Lolita Genomics
Jose Avila Cervantes
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DOI: dx.doi.org/10.17504/protocols.io.3byl4jb78lo5/v1
Protocol Citation: Jose Avila Cervantes 2023. DNA - Ball Python DNA Amplification with TFEC primers. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jb78lo5/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 18, 2022
Last Modified: March 27, 2023
Protocol Integer ID: 71489
Abstract
PCR amplification with Tfec set Exon 5 to genotype Piedball morph in Ball python (Python regius)

LEFT PRIMER AACTCAGAGCACTCCATGACC
RIGHT PRIMER CAGGTGTGCCCCTTTCATAA
REAGENTS
  • 10X PCR Buffer
  • MgSO4Concentration20 millimolar (mM)
  • Taq Polymerase 5U/ul
  • Primer Tfec exon5 Left Concentration10 micromolar (µM)
  • Primer Tfec exon5 Right Concentration10 micromolar (µM)
  • DNTP's Concentration10 micromolar (µM)
  • Ultra Pure Water
  • 100bp ladder (100-2,000 bp)
  • SYBR Safe DNA stain
  • Loading dye
  • Agarose

EQUIPMENT
  • DNA LoBind tubes 1.5mL
  • PCR 8-Strip tubes 0.2mL
  • Micropipettes
  • Thermal Cycler

MASTER MIX

Below is the recommended volume for one sample. Adjust for the number of samples to process and add 10% to account for pipetting error. Thaw all reagents at room temperature before using them except the Taq DNA polymerase, which has to be kept on ice at all times. Premix all reagents and add the Taq DNA polymerase at the end, vortex thoroughly for Duration00:00:30 seconds. Aliquot the master mix in to each tube Amount28 µL and then add Amount2 µL of DNA sample. The final volume for each reaction is Amount30 µL .
AB
ReagentVolume (uL)
10x PCR Buffer3
MgSO44.5
Taq Polymerase0.3
Primer F1.5
Primer R1.5
DNTP's0.6
Template-DNA2
Water16.6
Table 1. Master Mix for PCR.













30s
Mix
PCR CONDITIONS

ABCD
STEPTIMETEMPERATURE (ºC)
Initial Denaturation3 min94
Denaturation15 sec9425 Cycles
Annealing30 sec53.2
Extension30 sec74
Final Extension2 min74
Table 2. Thermocycler program



PCR
AMPLICON QUALITY CHECK

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

2. Add Amount4 µL of 100bp ladder (100-2,000 bp) in the first well.
3. Premix Amount4 µL of sample and Amount1 µL of 10X loading dye or Amount3 µL of sample and Amount1 µL of 6X loading dye. Load this mixture in each well.

3. Run the samples for Duration00:35:00 at a 100V.

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

5. The product should be a clear, single band around 200 bp.



35m
Analyze