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Team:Evry/Protocols
From 2012.igem.org
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<h1>Préparation of LB medium and LB Agar:</h1> | <h1>Préparation of LB medium and LB Agar:</h1> | ||
Revision as of 13:01, 3 August 2012
Contents |
PCR with Phusion High-Fidelity DNA Polymerase
Tube preparation
Put items in this order:
| Component | 50µl reaction | Comments |
| H2O | 32 | |
| 5x Phusion HF Buffer | 10 | |
| 10mM dNTPs | 1 | |
| Primer FW | 2 | Primers have to be at 10µM |
| Primer RV | 2 | Primers have to be at 10µM |
| Template DNA | 1 | |
| DMSO (optional) | 1,5 | recommended for GC-rich amplicons < 20kb |
| Phusion DNA polymerase | 0,5 |
Cycling instructions
| Cycle step | Temperature | Time | Cycles |
| Initial denaturation | 98°C | 4min | 1 |
| Denaturation | 98°C | 20s | 30 |
| Annealing | Lower Tm of primers | 30s | |
| Extension | 72°C | 30S/kb | |
| Final extension | 72°C | 10min | 1 |
| 4°C | hold |
Préparation of LB medium and LB Agar:
=> LB Agar :
-18,5g LB Agar
-300ml H2O
=> LB medium : -6g LB broth -300ml de H2O
Autoclaved at 250°C
