Team:Imperial College London/Wetlab/Protocols/PCR

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=PCR Protocol=
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{{Imperial/09/Tabs/Main/Wetlab/Protocols}}
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=PCR=
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PCR has three phases:
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*<b>Denaturation</b> - <i>1 cycle</i> at 95°C
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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.
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*<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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==Reaction Mixture ==
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===Aims===
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To amplify our DNA.
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Each PCR tube should contain:
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===Equipment===
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(25ul total)
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*PCR machine - we have a set programme - number <b>40</b>.
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* 2.5ul of 10x Pfu Ultra Buffer
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*Heating block
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* 0.5ul dNTPs
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*Eppendorf tubes
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* 1ul Primer Fwd (100ng)
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*P200, P10 and P2 Gilsons and tips
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* 1ul Primer Rev (100ng)
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* 0.5ul Pfu Ultra
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* 1ul DNA Template
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* 18.5ul ddH2O
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This can be made as a master mix, to reduce variation between reaction mixtures.
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Master Mix contents (for 4 PCR tubes):
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* 12.5ul Pfu Buffer
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* 2.5ul dNTPs
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* 5ul Primer Fwd (500ng)
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* 5ul Primer Rev (500ng)
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* 2.5ul Pfu Ultra
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* 92.5ul ddH2O
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Pipette 24ul of the master mix into different PCR tubes. To each experimental tube add 1ul DNA template, and to the negative control, add 1ul ddH2O.
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===Reagents===
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*10x Pfu Ultra buffer
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*dNTPs
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*Forward and Reverse primers
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*Pfu Ultra II
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*Template DNA
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== Cycles ==
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=Protocol=
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<i> X = Annealing Temperature - depending on primers (X1 temp without overhang, X2 temp with overhang), Y = Length of contruct to work out elongation time (for Pfu Ultra II, the rate is 15sec per kB, minimum 15 seconds) </i><br><br>
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<b><i>DNA Template</i></b>
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*Heat up heating block to 95°C.
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*Select distinct colonies from plate for testing.
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*Pipette 100ul sterile ddH<sub>2</sub>O into eppendorf tubes.
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*Pick each colony in turn, replica plate the colony on a fresh plate and then mix the loop tip with the water in the eppendorf tube to leave cells as a sample.
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*Boil the water and cells solution for 5 minutes in the heating block.
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*each template is now ready to be used as a DNA template in a PCR reaction.
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1 cycle: 2 minutes at 95'C <br>
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<b><i>PCR Reaction</i></b><br>
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10 cycle: 30 seconds at 95'C, 45 seconds at X'C, Y seconds at 72'C <br>
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<b>Reaction Mixture</b><br>
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20 cycle: 30 seconds at 95'C, 45 seconds at X2'C, Y seconds at 72'C <br>
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<i>25ul total volume</i>
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1 cycle: 5 minutes at 72'C <br>
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*2.5ul 10xPfu Ultra Buffer
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*1ul Primer Fwd (100ng)
 +
*1ul Primer Rev (100ng)
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*0.5ul Pfu Ultra
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*1ul DNA template
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== Tips ==
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<i>TIP:</i> For repeats of the <b>SAME</b> 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 ddH<sub>2</sub>O. Make up your positive control separately).
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* Use master mixes. They reduce the variation between reaction mixtures.
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* Negative control has no DNA template in it.
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<b>Cycles</b><br>
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* Vary the first annealing temperature across a gradient (usually in 2'C intervals), and keep the second annealing temperature constant. The temperature of the negative should always be at the lowest temperature of the reaction.
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<i>X=annealing temperature</i>. 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).<br>
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<i>X2=annealing temperature 2</i>. 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.<br>
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<i>Y=Time</i> (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.<br>
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<br>
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*1 cycle - 2 minutes at 95°C
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*10 cycles - 30 secs at 95°C, 45 secs at X, Y secs at 72°C
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*20 cycles - 30 secs at 95°C, 45 secs at X2, Y secs at 72°C
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*1 cycle - 5 minutes at 72°C
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Latest revision as of 13:09, 2 October 2009



Contents

PCR

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 - we have a set programme - number 40.
  • Heating block
  • Eppendorf tubes
  • P200, P10 and P2 Gilsons and tips

Reagents

  • 10x Pfu Ultra buffer
  • dNTPs
  • Forward and Reverse primers
  • Pfu Ultra II
  • 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 the loop tip with the water 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: 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).

Cycles
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

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