Nov 19, 2020
High GC-content PCR
- 1Ronin Institute
Protocol Citation: Joao Vitor Molino 2020. High GC-content PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bprimm4e
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: November 16, 2020
Last Modified: November 19, 2020
Protocol Integer ID: 44554
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Abstract
X7 is a a Pfu-sso7d fusion DNA polymerase that is compatible with uracil-excision cloning and has been used with sucess to amplify our high GC-content sequences and it is a valuable tool for USER clonning.
Before starting
Please note that protocols with X7 may differ from protocols with other polymerases. Conditions recommended below were tested with our sequences, mostly high GC-content sequences from Chlamydomonas reinhardtii expression vectors.
Please refer to the original work that develop the fusion polymerase.
All components should be mixed prior to use.
Reference:
Nørholm, M. H. H. (2010). A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology, 10. https://doi.org/10.1186/1472-6750-10-21
Materials
- Phusion® GC Buffer (NEB) 5X
- Betaine 5M
- dNTPs 10 uM
- 10 µM Forward Primer
- 10 µM Forward Primer
- Template DNA
- X7 polymerase
- Nuclease-Free Water
- Ice bucket
- Thermocycler
Reaction preparation
Reaction preparation
Set up the reaction using the following table on ice:
| Reagent | Volume (uL) | |
| Phusion® GC Buffer (NEB) 5X | 10 | |
| Betaine 5M | 10 | |
| dNTPs 10 uM | 1 | |
| 10 µM Forward Primer | 2.5 | |
| 10 µM Forward Primer | 2.5 | |
| Template DNA | Variable (~0.5-1) | |
| X7 | 0.5 | |
| Nuclease-Free Water | 23 | |
| Final Volume | 50 |
X7 polymerase develop in Nørholm, M. H. H. (2010). A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology, 10. https://doi.org/10.1186/1472-6750-10-21
Mix the reaction gently and spin it down if necessary.
Transfer PCR tubes to a thermocycler and start the PCR program. Below it is presented a default setup which can varied accordingly to the target amplicon. The recommended cycle setup and temperatures are shown below.
| STEP | TEMP | TIME | |
| Initial Denaturation | 98 °C | 30 seconds | |
| 35-40 Cycles | 98 °C | 10 seconds | |
| 60 °C | 20 seconds | ||
| 68 °C | 1 minute/kb | ||
| Final Extension | 72 °C | 5 minutes | |
| Hold | 4 – 10 °C |
Melting temperature can be adjusted accordingly with the primers used.