Team:HKUST/Protocols/PCR

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(Difference between revisions)
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3. Vortex and then spin down.<br>
3. Vortex and then spin down.<br>
4. Put it into the PCR machine and set the program.<br>
4. Put it into the PCR machine and set the program.<br>
-
    Program<br>
+
    &nbsp; Program<br>
-
     (1) Initial denaturation  95 °C  4 mins<br>
+
     &nbsp;  &nbsp;  (1) Initial denaturation  95 °C  4 mins<br>
-
     (2) Run 25-30 cycles of:<br>
+
     &nbsp;  &nbsp;  (2) Run 25-30 cycles of:<br>
-
        Denaturation           95 °C  30 secs<br>
+
        &nbsp;  &nbsp;  Denaturation   &nbsp;          95 °C  30 secs<br>
-
        Annealing                     30 secs <br>
+
      &nbsp;  &nbsp;    Annealing       &nbsp;        &nbsp;          30 secs <br>
-
                                Temperature is depended on melting temperature of primer. <br>
+
                &nbsp;  &nbsp;      &nbsp;  Temperature is depended on melting temperature of primer. <br>
-
                                Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br>
+
                    &nbsp;  &nbsp;  &nbsp;      Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent<br>
-
        Extension              72 °C  30 secs per 500bp PCR product length<br>
+
      &nbsp;  &nbsp;    Extension              72 °C  30 secs per 500bp PCR product length<br>
-
     (3) Final extension       72 °C  3~5 mins<br>
+
     (3) Final extension     &nbsp;    72 °C  3~5 mins<br>
<p> Tips: </p>
<p> Tips: </p>
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.
The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.

Revision as of 15:32, 19 October 2009

Salt and Soap template

a

PCR

Purpose: To amplify a specific piece of DNA out from the whole.

Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent. Taq gives sticky ends after PCR, while Vent gives blunt ends.

Procedure:

1. Add 10 μL water to make it a 20 μL reaction.
2. Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase
3. Vortex and then spin down.
4. Put it into the PCR machine and set the program.
  Program
    (1) Initial denaturation 95 °C 4 mins
    (2) Run 25-30 cycles of:
    Denaturation   95 °C 30 secs
    Annealing     30 secs
      Temperature is depended on melting temperature of primer.
      Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent
    Extension 72 °C 30 secs per 500bp PCR product length
(3) Final extension   72 °C 3~5 mins

Tips:

The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.
HKUST