IGEM:IMPERIAL/2009/PCR Protocol: Difference between revisions
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*<b>Annealing</b> - <i>10 cycles</i> - this is the '<i>seeding</i>' phase; it occurs without binding of the XbaI and SpeI regions on the primers when binding the template DNA. | *<b>Annealing</b> - <i>10 cycles</i> - this is the '<i>seeding</i>' phase; it occurs without binding of the XbaI and SpeI regions on the primers when binding the template DNA. | ||
*<b>Elongation</b> - <i>20 cycles</i> - this is where the final end product comes from - all regions of the primers bind at this stage. | *<b>Elongation</b> - <i>20 cycles</i> - this is where the final end product comes from - all regions of the primers bind at this stage. | ||
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===Aims=== | ===Aims=== | ||
To amplify our DNA. | To amplify our DNA. |
Revision as of 08:01, 12 August 2009
PCR has three phases:
- Denaturation - 1 cycle at 95°C
- Annealing - 10 cycles - this is the 'seeding' phase; it occurs without binding of the XbaI and SpeI regions on the primers when binding the template DNA.
- Elongation - 20 cycles - this is where the final end product comes from - all regions of the primers bind at this stage.
Aims
To amplify our DNA.
Equipment
- PCR machine
- Heating block
- Eppendorf tubes
- P200, P10 and P2 Gilsons and tips
Reagents
- 10x Pfu Ultra buffer
- dNTPs
- Forward and Reverse primers
- Pfu Ultra
- Template DNA
Protocol
DNA Template
- Heat up heating block to 95°C.
- Select distinct colonies from plate for testing.
- Pipette 100ul sterile ddH2O into eppendorf tubes.
- Pick each colony in turn, replica plate the colony on a fresh plate and then mix th loop tip with thewater in the eppendorf tube to leave cells as a sample.
- Boil the water and cells solution for 5 minutes in the heating block.
- each template is now ready to be used as a DNA template in a PCR reaction.
PCR Reaction Reaction Mixture 25ul total volume
- 2.5ul 10xPfu Ultra Buffer
- 1ul Primer Fwd (100ng)
- 1ul Primer Rev (100ng)
- 0.5ul Pfu Ultra
- 1ul DNA template