Revision as of 12:53, 1 July 2008

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The protocols we used

(estimated time: 3 hours and 30 min)

Materials needed:

For every DNA sample you want to amplify, put:
- 2 µl buffer
- 0.6 µl MgCl2
- 0.4 µl dNTPs
- 1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
- 0.2 µl Taq Polymerase
- 250 nM VF2 primer
- 250 nM VR primer
- A proper amount of ddH2O to have 20 µl of total reaction volume
into an eppendorf tube.
Put the eppendorf tube in the thermal cycler and set this program:
- 95°C 10 min
- CYCLE:
  - 95°C 30 sec
  - 60°C 1 min
  - 72°C 1 min
- for 35 cycles (40 if you are performing a colony PCR)
- 72°C 7 min
- 16°C forever.
Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.

@@ Line 22: / Line 22: @@
 *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
 *[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
+*[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]]
 *[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
 *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]