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.
To amplify our DNA.
- PCR machine - we have a set programme - number 40.
- Heating block
- Eppendorf tubes
- P200, P10 and P2 Gilsons and tips
- 10x Pfu Ultra buffer
- Forward and Reverse primers
- Pfu Ultra II
- Template DNA
- 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.
25ul total volume
- 2.5ul 10xPfu Ultra Buffer
- 1ul Primer Fwd (100ng)
- 1ul Primer Rev (100ng)
- 0.5ul Pfu Ultra
- 1ul DNA template
TIP: For repeats of the SAME DNA - make up a 'master mix' - multiply the above volumes by the number of samples you have (excluding the DNA for each). Then divide this master mix between your eppendorf tubes and add your DNA (REMEMBER: you will also need a positive and negative control. Your negative control will not contain any DNA - make this up to the total volume with ddH2O. Make up your positive control separately).
X=annealing temperature. This is varied at 2°C intervals. The temperature is decided using the melting temperatures of the primers as top temperature (this will become more intuitive the more you do the procedure).
X2=annealing temperature 2. This is kept constant. The temperature is decided using the melting temperatures of the primers including the sequence for XBaI and SpeI. X2 must be slightly below the predicted temperature.
Y=Time (in seconds). This is determined using the length of the construct to calculate the elongation time. We're using Pfu Ultra II which works at 15 seconds per kb. Never do less than 15 seconds.
- 1 cycle - 2 minutes at 95°C
- 10 cycles - 30 secs at 95°C, 45 secs at X, Y secs at 72°C
- 20 cycles - 30 secs at 95°C, 45 secs at X2, Y secs at 72°C
- 1 cycle - 5 minutes at 72°C