May 15, 2025
Fast-COLD PCR Protocol for Enrichment of TP53 R181H Variant Before Sanger Sequencing
- Catalin V. Munteanu1
- 1Victor Babes University of Medicine and Pharmacy Timisoara
- Molpath
Protocol Citation: Catalin V. Munteanu 2025. Fast-COLD PCR Protocol for Enrichment of TP53 R181H Variant Before Sanger Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1mbr7vr2/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: May 15, 2025
Last Modified: May 15, 2025
Protocol Integer ID: 218269
Keywords: mosaic tp53 r181h variant, cold pcr protocol for enrichment, tp53, cold pcr program before sanger, cold pcr protocol, cold pcr program, pcr, lab steps for enrichment, pcr protocol
Abstract
This protocol describes the wet-lab steps for enrichment of mosaic TP53 R181H variant using Fast-COLD PCR program before Sanger sequencing.
Materials
Reagents:
- AmpliTaq Gold™ 360 Master Mix 1 mL, Applied Biosystems™, Catalog #4398876
- DNase/RNase-Free Water
- HPLC-purified oligos (Fwd and Rev primers)
Equipments:
- GeneAmp™ PCR System 9700 Fast Thermal Cycler
Primer sequences:
- Fwd: 5’-ACCAGCCCTGTCGTCTCT-3’
- Rev: 5’-GTGCAGCTGTGGGTTGATTC-3’
Troubleshooting
Fast-COLD PCR Protocol
Reagents/reaction tube:
12.5 µL AmpliTaq Gold™ 360 Master Mix
8.5 µL DNase/RNase-Free Water
2 µL 360 GC Enhancer
1 µL DNA template (10 – 50 ng/µL)
0.5 µL Fwd oligos (10 µM)
0.5 µL Rev oligos (10 µM)
Thermocycler program:
Initial denaturation: 95°C, 10 min
First 15 cycles: Denaturation: 95°C, 30 s; Annealing: 59°C, 30 s; Extension: 72°C, 30 s
Last 20 cycles: Denaturation: 87°C, 30 s; Annealing: 59°C, 30 s; Extension: 72°C, 30 s
Final extension: 72°C, 7 min