Jul 07, 2020

Public workspaceAFLP RedTaq protocol

DOI
dx.doi.org/10.17504/protocols.io.rpvd5n6
  • 1W. Szafer Institute of Botany, Polish Academy of Sciences
  • Molecular Biogeography Group
Tomasz Suchan
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DOI: dx.doi.org/10.17504/protocols.io.rpvd5n6
Protocol Citation: Michal Ronikier, Tomasz Suchan 2020. AFLP RedTaq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rpvd5n6
Manuscript citation:
Paun, O., & Schönswetter, P. (2012). Amplified fragment length polymorphism: an invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies. In Plant DNA Fingerprinting and Barcoding (pp. 75-87). Humana Press.
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: July 16, 2018
Last Modified: July 07, 2020
Protocol Integer ID: 13781
Materials
MATERIALS
ReagentEcoRI - 10,000 unitsNew England BiolabsCatalog #R0101S
ReagentMseI - 500 unitsNew England BiolabsCatalog #R0525S
ReagentBSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S
ReagentWater, nuclease free
ReagentNaCl 0.5 M
ReagentT4 DNA Ligase (5U/uL)RocheCatalog #10 799 009 001
ReagentREDTaq DNA Polymerase (1U/uL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D4309
ReagentdNTP mix (2.5 mM each)
Before start
Dilute BSA to 1 mg/ml
Oligonucleotides used:
EcoR1-Adapters 5-CTCGTAGACTGCGTACC 5-AATTGGTACGCAGTCTAC final: 5 μM of each MseI-Adapters 5-GACGATGAGTCCTGAG 5-TACTCAGGACTCAT final: 50 μM of each
Presel Primer Eco: gactgcgtaccaattca final: 5 μM of each Presel Primer Mse: gatgagtcctgagtaac final: 5 μM of each
Eco-Primer gac tgc gta cca att cxx x final: 1 μM Mse Primer gat gag tcc tga gta axx x (FAM-6 labeled) final: 5 μM
Prepare Eco adapters at 5 µM and Mse adapters at 50 µM (working solutions):
Adapter E1  stock (100 µM)10 µl
Adapter E2 stock (100 µM)10 µl
ddH2O180 µl
Adapter M1 stock (100 µM)100 µl
Adapter M2 stock (100 µM)100 µl
ddH2O0 µl
Incubate for 5 min at 95°C on the thermoblock/thermocycler and let it cool down slowly; eg. at 37°C on thermoblock/thermocycler.
Restriction-ligation reaction
Restriction-ligation reaction
Amount0.53 µL water
Amount1.2 µL T4 DNA Ligase buffer (10×)
Amount0.6 µL BSA (1 mg/ml)
Amount1.2 µL NaCl (0.5 M)
Amount1 µL Adapter MseI (50 µM)
Amount1 µL Adapter EcoRI (5 µM)
Amount0.1 µL MseI (10 U/µl)
Amount0.25 µL EcoRI (20 U/µl)
Amount0.12 µL T4 DNA Ligase (5U/µl)
Add 6 μl of DNA to 6 μl of the restriction-ligation mix.
Incubate at 37ºC for 3 h and at 17 °C overnight.
Dilute ligated DNA fragments 10-fold.
Preselective PCR
Preselective PCR
Prepare the reaction mix:
Amount6.09 µL water
Amount1.14 µL RedTaq Buffer (10x)
Amount0.22 µL dNTP (2.5 mM each)
Amount0.15 µL Presel. primer Eco-A (10 µM)
Amount0.15 µL Presel. primer Mse-C (10 µM)
Amount0.25 µL Sigma RedTaq (1U/ µl)
Add 2 μl of the 10x diluted restriction-ligation product to 8 μl of the reaction mix (total volume 10 μl).
Run the PCR program:
  • 72°C for 2 min
  • 25 cycles of:
  • 94°C for 1 s
  • 56°C for 30 s
  • 72°C for 120 s
  • 60°C for 30 min
  • hold at 4°C
Dilute the PCR product 10-fold.
Selective PCR
Selective PCR
Prepare reaction mix:
Amount5.45 µL water
Amount1 µL RedTaq Buffer (10x)
Amount0.22 µL dNTP (2.5 mM each)
Amount0.25 µL Sigma RedTaq (1U/ µl)
Amount0.54 µL Sel. primer Mse-Cxx (5 µM)
Amount0.54 µL Sel. primer Eco-Axx (1 µM)
Add 2 μl of the 10x diluted preselective PCR product to 8 μl of the reaction mix (total volume 10 μl).
Run the PCR program:
  • 94°C for 2 min
  • 10 cycles of:
  • 94°C for 1 s
  • 65°C for 30 s, with a ramp of -1°C per step (up to 56°C)
  • 72°C for 120 s
  • 22 cycles of:
  • 94°C for 1 s
  • 56°C for 30 s
  • 72°C for 120 s
  • 60°C for 30 min
  • hold at 4°C
Purification
Purification
Proceed with Sephadex purification protocol and loading on ABI 3130.