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Colony PCR
From 2011.igem.org
(Difference between revisions)
| Line 39: | Line 39: | ||
2. 94°C - 30 sec | 2. 94°C - 30 sec | ||
| - | 51°C - 1.00 min | + | 3. 51°C - 1.00 min |
| - | 72°C - 3.00 min | + | 4. 72°C - 3.00 min |
| - | repeat | + | repeat 2-4 for 40 cycles |
| - | 72°C - 10.00 min | + | 5. 72°C - 10.00 min |
| - | 4°C - forever | + | 6. 4°C - forever |
[https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back'''] | [https://2011.igem.org/Team:Panama/Protocols/Wetlab '''Back'''] | ||
Latest revision as of 02:47, 29 September 2011
Materials:
Agar plate with colonies
PCR mastermix
Forward primers
Reverse primers
Molecular grade H2O
agar plate with appropriate antibiotic
Procedure:
1. Prepare a stock solution using per colony
12.5ul mastermix
2.5ul forward primer
2.5ul reverse primer
7.5ul Molecular grade H2O
2. aliquot in 0.6 ml PCR tubes 25ul of the stock solution. Do this for every colony you intend to pick.
3. using a micropipette with a yellow tip tap the colony you want to pick and dip the tip in one of the PCR tubes.
4. streak the colony onto an agar plate to save the colony.
5. run your PCR program.
we used:
1. 94°C - 5.00 min
2. 94°C - 30 sec
3. 51°C - 1.00 min
4. 72°C - 3.00 min
repeat 2-4 for 40 cycles
5. 72°C - 10.00 min
6. 4°C - forever
