From 2012.igem.org
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I. PCR
- Mix according to the following table, where 2 = LacZ, 3 = CAT*, 4 = tetr, 0.1-0.3 = +template, and 0.4 = -template
- For example, 2.1 = lacZ sample, 2.4 = lacZ control
| | 2.1 | 2.2 | 2.3 | 2.4 | 3.1 | 3.2 | 3.3 | 3.4 | 4.1 | 4.2 | 4.3 | 4.4
|
| Reagent | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) | (uL)
|
| water | 33.5 | 33.5 | 33.5 | 33.5 | 33.5 | 33.5 | 33.5 | 33.5 | 32.5 | 32.5 | 32.5 | 33.5
|
| 5x Phusion HiFi buffer | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10
|
| 10 mM dNTP mix | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1
|
| 10 uM primer (F) | 2.5 (p2f_2) | 2.5 (p2f_2 | 2.5 (p2f_2) | 2.5 (p2f_2) | 2.5 (p3f_2) | 2.5 (p3f_2) | 2.5 (p3f_2) | 2.5 (p3f_2) | 2.5 (p4f) | 2.5 (p4f) | 2.5 (p4f) | 2.5 (p4f)
|
| 10 uM primer (R) | 2.5 (p2r_2) | 2.5 (p2r_2) | 2.5 (p2r_2) | 2.5 (p2r_2) | 2.5 (p3r) | 2.5 (p3r) | 2.5 (p3r) | 2.5 (p3r) | 2.5 (p4r_2) | 2.5 (p4r_2) | 2.5 (p4r_2) | 2.5 (p4r_2)
|
| Template | ECNR2 colony* | ECNR2 colony* | ECNR2 colony* | 0 | ECFI5 colony* | ECFI5 colony* | ECFI5 colony* | 0 | 1 (pBR322) | 1 (pBR322) | 1 (pBR322) | 0
|
| Phusion HiFi polymerase | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5
|
*For these templates, I touched a colony with a sterile pipette tip and spread it on the bottom of the PCR tube before adding reagents
| Step | Temp (oC) | Time
|
| 1 | 94 | 4 m
|
| 2 | 94 | 30 s
|
| 3 | 53 | 30 s
|
| 4 | 72 | 1.5 m
|
| 5 | GOTO 2 | 7x
|
| 6 | 94 | 30 s
|
| 7 | 66 | 30 s
|
| 8 | 72 | 1.5 m
|
| 9 | GOTO 6 | 30x
|
| 10 | 72 | 5 m
|
| 11 | 4 | ∞*
|
- Store tubes on ice after PCR is finished
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