Team:Newcastle/PCR

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(Difference between revisions)
(Materials required)
(Materials required)
Line 8: Line 8:
# 32.5 µl of distilled H<sub>2</sub>O
# 32.5 µl of distilled H<sub>2</sub>O
# 10 µl of 5x GoTaq Buffer
# 10 µl of 5x GoTaq Buffer
-
# 1 µl of nucleotide dNTPs
+
# 1 µl of dNTPs
# 2.5 µl forward primer
# 2.5 µl forward primer
# 2.5 µl backward primer
# 2.5 µl backward primer
# 1 µl template DNA
# 1 µl template DNA
 +
# 0.5 µl of GoTaq polymerase
==Conditions for ThermoCycler==
==Conditions for ThermoCycler==

Revision as of 13:15, 29 July 2010

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Contents

GoTaq PCR

Materials required

Add the following as mentioned below:

  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

  1. Initialise - 95°C for 2 minutes.
  2. Denature - 95°C for 30 seconds.
  3. Anneal - 52°C for 30 seconds (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 according:

  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

  1. Initialise - 98°C for 30 seconds.
  2. Denature - 98°C for 10 seconds.
  3. Anneal - x°C for 20 seconds (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.

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