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Team:TU Darmstadt/Notebook/Methods/PCR with Pfu polymerase
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href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_ligation" >DNA ligation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_quantification_with_NanoDrop" >DNA quantification with NanoDrop</a></li><li"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_purification" >PCR purification</a></li><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_with_Pfu_polymerase" >PCR with Pfu polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Plasmid_preparation" >Plasmid preparation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Restriction_digest" >Restriction digest</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/SDS-PAGE" >SDS-PAGE</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Bacterial_cell_culture" >Bacterial cell culture</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Cell_counting_plating" >Cell counting/plating</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Chemically_competent_cells" >Chemically competent cells</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Colony_PCR_with_Taq_polymerase" >Colony PCR with Taq polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Glycerine_stocks" >Glycerine stocks</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Heat_shock_transformation" >Heat shock transformation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Protein expression" >Protein expression</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/hydrochloric_acid_extraction" >37% hydrochloric acid extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dichloromethane_extraction" >Dichloromethane extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Methanol_extraction" >Methanol extraction</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Ethanol_extraction" >Ethanol extraction</a></li></ul></li></ul></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Team" >Team</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Gallery" >Gallery</a></li></ul> |
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Revision as of 01:06, 16 October 2014
PCR with Pfu polymerase
Short explanation:
PCR (polymerase chain reaction) is a method for exponentially amplifying a fragment of DNA in vitro.
Equipment:
- Micropipettes with sterile tips
- PCR machine
- PCR tubes
Chemicals & consumables: 50 µL batch
- 18 µL Pfu Mastermix 9:25
- 100 ng = 1 µL DNA template
- 1 µL Forward primer (10 µM)
- 1 µL Reverse primer (10 µM)
- up to 50 µL ddH2O
Procedure:
Standard protocol for Pfu Polymerasep>
| # | Temperature | Time | ||
| 1 | 95°C | 00:05:00 | ||
| 2 | 95°C | 00:00:20 | ||
| 3 | 55°C | 00:00:30 | ||
| 4 | 72°C | 2 min/kbp | ||
| 5 | GO TO 2 | REPEAT 30x | ||
| 6 | 72°C | 3 min/kbp | ||
| 7 | 4°C | HOLD |
