Oct 05, 2021
Version 6
Polymerase chain reaction (PCR) V.6
- 12021 iDEC NEFU_China
- NEFU_China 2021
Protocol Citation: Shuning Guo 2021. Polymerase chain reaction (PCR). protocols.io https://dx.doi.org/10.17504/protocols.io.bysrpwd6Version created by Shuning Guo
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 05, 2021
Last Modified: October 05, 2021
Protocol Integer ID: 53809
Abstract
This protocol is used to amplify target DNA fragment for plasmid construction or other use.
Materials
2×High Fidelity Master Mix (MCLAB)/2×Rapid Master Mix (Vazyme)
ddH2O
Template
F/R Primer
Thermo cycler.
Safety warnings
Please wear gloves for the experiment, don't try to touch the lid after PCR program initiation.
Before start
Set up a small box with ice, put DNA and 2×high Fidelity Master Mix (MCLAB)/or 2×Rapid Master Mix (Vazyme) into it before going into the thermocycler.
Choose one case from the cases below.
Simple PCR for amplifying target DNA fragments5 steps
Add the following reagent to a PCR tube.(50 μl).
| A | B | |
| 2×High Fidelity Master Mix (MCLAB) | 25 μl | |
| Template | 1 μl | |
| Forward Primer (10 μM) | 1 μl | |
| Reverse Primer (10 μM) | 1 μl | |
| ddH2O | 22 μl |
Program the thermocycler as follows:
| A | B | |
| Temperature | Time | |
| 95/98℃ | 5 min | |
| 95/98℃ | 30 s | |
| Tm-3~5℃ | 30 s | |
| 72℃ | 1kbp/min | |
| 72℃ | 5~10 min | |
| 16℃ | ∞ |
Repeat 30 times in 3-5 steps
Use the palm centrifuge to mix the solution in PCR tube.
Put the PCR tube into the thermocycler and Run the program.
Using agarose gel electrophoresis to confirm if correct construct was present.