IGEM:IMPERIAL/2009/PCR Protocol: Difference between revisions
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*Heat up heating block to 95°C. | *Heat up heating block to 95°C. | ||
*Select distinct colonies from plate for testing. | *Select distinct colonies from plate for testing. | ||
*Pipette 100ul sterile ddH< | *Pipette 100ul sterile ddH<sub>2</sub>O 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. | *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. | *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. | *each template is now ready to be used as a DNA template in a PCR reaction. | ||
<b><i>PCR Reaction</i></b> | <b><i>PCR Reaction</i></b><br> | ||
<b>Reaction Mixture</b> | <b>Reaction Mixture</b><br> | ||
<i>25ul total volume</i> | <i>25ul total volume</i> | ||
*2.5ul 10xPfu Ultra Buffer | *2.5ul 10xPfu Ultra Buffer | ||
| Line 38: | Line 38: | ||
*0.5ul Pfu Ultra | *0.5ul Pfu Ultra | ||
*1ul DNA template | *1ul DNA template | ||
<i>TIP:</i> 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 ddH<sub>2</sub>O). | |||
Revision as of 08:06, 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
TIP: 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).
