IGEM:IMPERIAL/2009/PCR Protocol

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