Team:Newcastle/PCR

From 2010.igem.org

(Difference between revisions)
(Conditions for ThermoCycler)
(Conditions for ThermoCycler)
Line 45: Line 45:
# Initialise - 98°C for 30 seconds.
# Initialise - 98°C for 30 seconds.
# Denature - 98°C for 10 seconds.
# Denature - 98°C for 10 seconds.
-
# Anneal - x°C for 20 seconds (melting temperature, Tm, of template)
+
# Anneal - x°C for 20 seconds (depends upon the melting temperature, Tm, of template)
# Extension - 72°C for 30 seconds per kb
# Extension - 72°C for 30 seconds per kb
# Extension finish - 72°C for 5-10 minutes
# Extension finish - 72°C for 5-10 minutes

Revision as of 13:35, 29 July 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Contents

GoTaq PCR

Materials required

Add the following as mentioned below to make up to a final volume of 50µl in the PCR tube:

  1. 32.5 µl of distilled H2O
  2. 10 µl of 5x GoTaq Buffer
  3. 1 µl of dNTPs
  4. 2.5 µl forward primer
  5. 2.5 µl backward primer
  6. 1 µl template DNA
  7. 0.5 µl of GoTaq polymerase

Conditions for ThermoCycler

After putting the PCR tubes in the thermocycler, set the thermocycler's condition as mentioned below:

  1. Initialise - 95°C for 2 minutes.
  2. Denature - 95°C for 30 seconds.
  3. Anneal - 52°C for 30 seconds (depends upon the melting temperature, Tm, of template)
  4. Extension - 75°C for 30 seconds
  5. Extension finish - 75°C for 5 minutes
  6. Hold - 4°C

Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.

Phusion PCR

Materials required

Add the following as mentioned below to make up to a final volume of 50µl in the PCR tube:

  1. 27.5 µl of distilled H2O
  2. 10 µl of 5x Buffer
  3. 1 µl of dNTPs
  4. 5 µl forward primer
  5. 5 µl backward primer
  6. 1 µl template DNA
  7. 0.5 µl of Fusion

Conditions for ThermoCycler

After putting the PCR tubes in the thermocycler, set the thermocycler's condition as mentioned below:

  1. Initialise - 98°C for 30 seconds.
  2. Denature - 98°C for 10 seconds.
  3. Anneal - x°C for 20 seconds (depends upon the melting temperature, Tm, of template)
  4. Extension - 72°C for 30 seconds per kb
  5. Extension finish - 72°C for 5-10 minutes
  6. Hold - 4°C

Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.

Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon