From 2013.igem.org
30-09 - 06-10-13
Generation of DelH Plasmid pHM04 15-09
Colony-PCR Conditions CP.W23.A
| Template | 23x 1 picked colony, positive control
|
| Expected length [bp] | 663
|
| Named | 1A - 12H
|
| Primer fw 10 µM | 100x 2 µl VF2
|
| Primer rev 10 µM | 100x 2 µl DN07
|
| iTaq Polymerase (2x) | 100x 10 µl
|
| ddH2O | 96x 6 µl
|
| Cycles | Temperature DelH [°C] | Time [s]
|
| 1 | 95 | 120
|
| 12 | 95 | 30
|
| 68 (touchdown -0.5°C) | 30
|
| 72 | 45
|
| 18 | 95 | 30
|
| 65 | 30
|
| 72 | 45
|
| 1 | 12 | inf
|
Result
Expected band: 663 bp
[[|200px|thumb|right|Fig.23.1 Colony PCR of Gibson assembled DelH-BB without mRFP 23 colonies]]
Amplification of backbone pSB6A1 for strategy pFS02
| Reagent | Amount
|
| Template | 1 µl of minipreped pSB6A1 (2L)
|
| Primer fw 10 µM | 2 µl FS78
|
| Primer rev 10 µM | 2 µl HM17
|
| Phusion Flash Ready Mix | 10 µl
|
| ddH2O | 5 µl
|
| Cycles | Temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 (touchdown -0,5°C) | 5
|
| 72 | 1:25
|
| 18 | 98 | 1
|
| 68 | 5
|
| 72 | 1:25
|
| 1 | 72 | 5 min
|
| 1 | 4 | inf
|
| Cycles | Temperature [°C] | Time [s]
|
| 1 | 98 | 5
|
| 12 | 98 | 1
|
| 58 | 5
|
| 72 | 1:25
|
| 1 | 72 | 5 min
|
| 1 | 4 | inf
|
Amplification of backbone pSB6A1 for strategy pFS02
| Reagent | Amount
|
| Template | 1 µl of minipreped pSB6A1 (2L)
|
| Primer fw 10 µM | 2 µl FS85
|
| Primer rev 10 µM | 2 µl HM17
|
| Phusion Flash Ready Mix | 10 µl
|
| ddH2O | 5 µl
|
| Cycles | Temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 (touchdown -0,5°C) | 5
|
| 72 | 1:25
|
| 18 | 98 | 1
|
| 68 | 5
|
| 72 | 1:25
|
| 1 | 72 | 5 min
|
| 1 | 4 | inf
|
| Cycles | Temperature [°C] | Time [s]
|
| 1 | 98 | 5
|
| 12 | 98 | 1
|
| 58 | 5
|
| 72 | 1:25
|
| 1 | 72 | 5 min
|
| 1 | 4 | inf
|
"
