green light receptor
PCR to amplify Green light Promotor PcpcG
Investigators:Julia
Testdigest of quickchanged CcaR
Investigators:Julia,with help of the team
blue light receptor
3A-assembly
Investigators: Sandra
Digestion I:
- Lovtap
- Not-Gate
- pSB1A3
- pSB1T3
stored in the blue light box.
Ligation
- ♥-A3 Not
- ♥-A3 nOt
- ♥-A3 noT
- ♥-T3 Not
- ♥-T3 nOt
- ♥-T3 noT
stored in the ligation box.
Digestion II:
Also digestion with new amplified vector
Lovtap was digested again this time also with DpnI. So for this ligation the ♥-PCR3A, digested with DpnI, and pSB1C3 (new amplified with PCR, already digested with DpnI) and Not, nOt, noT were used.
- ♥-C3 Not
- ♥-C3 nOt
- ♥-C3 noT
Transformation
Investigators: Sophie
transformation of:
- ♥-A3 Not
- ♥-A3 nOt
- ♥-A3 noT
- ♥-T3 Not
- ♥-T3 nOt
- ♥-T3 noT
Incubation over night at 37°C.
PCR
Investigator: Sophie
PCR
| Name:
| Date:
|
| Continue from Experiment: 12.08.2011
Order primers
|
| Project Name:
more NOT-gate with mutated restriction site (NHE I instead of Spe I)
|
PCR-Mixture for one Reaction:
For a 50 µl reaction use
P24
P79
| 32,5µl
| H20
| Name
|
| 10µl
| 5x Phusion Buffer
| of Primer
|
| 2.5µl
| Primer fw
| P 24 (1:10)
|
| 2.5µl
| Primer dw
| P 79 (1:10)
|
| 1µl
| dNTPs
| of Template DNA
|
| 1µl
| DNA-Template
|
M45
|
| 0.5 µl
| Phusion (add in the end)
|
|
The program we used: RT 2step (see last PCR of NOT-gate)
| Temp.
| Time
| Nr. of cycles
|
| 94°
| 5’
| 1x
|
| 94°
| 30’’
| 2x
|
| 64°
| 30’’
|
| 72°
| 1’30’’
|
| 94°
| 30’’
| 2x
|
| 62°
| 30’’
|
| 72°
| 1’30’’
|
| 94°
| 30’’
| 5x
|
| 61,6°
| 30’’
|
| 72°
| 1’30’’
|
| 94°
| 30’’
| 25x
|
| 57,8°
| 30’’
|
| 72°
| 1’30’’
|
| 72°
| 7’
| 1x
|
| 4°
| ∞
|
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
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