green light receptor
PCR
Investigator: Jakob
PCR
| Name:
Jakob
| Date:
16.08.2011
|
| Continue from Experiment: 12.08.2011
Order primers
|
| Project Name:
Quickchange of CcaR (EcoRI)
|
PCR-Mixture for one Reaction:
For a 50 µl reaction use
P77:
- CACAGGAAACCtcactaATGAGgATcCTTTTAGTGGAGGATGATTTGCc
P78:
- gGCAAATCATCCTCCACTAAAAGgATcCTCATtagtgaGGTTTCCTGTG
| 32,5µl
| H20
| Name
|
| 10µl
| 5x Phusion Buffer
| of Primer
|
| 2.5µl
| Primer fw
| P 77 (1:10)
|
| 2.5µl
| Primer dw
| P 78 (1:10)
|
| 1µl
| dNTPs
| of Template DNA
|
| 1µl
| DNA-Template
| CcaR from synechocystis 5ng/µl
|
| 0.5 µl
| Phusion (add in the end)
|
|
The program we used: for Quickchange of CcaR (EcoRI)
| 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°
| ∞
|
continuing with quickchanged CcaS and CcaR
Investigator: Julia
on the plates for CcaR were no colonies at all :(
the quickchanged CcaS had about six colonies per plate.
nine were picked for miniprep ( 3ml LB medium with Kanamycin).
blue light receptor
Gel purification
Investigators:Sophie
the bands of LOV-pcr and NOT-pcr where purificated. The vector was digested again because the band on the gel was not intense enough.
Ligation
Investigators: Sophie:
LOV-pcr, NOT-pcr and the Amp vector where ligated for 1h at room temperature
Transformation
Investigators: Sophie:
Transformation with the ligation product
red light receptor
PCR
Investigator: Jakob
PCR
| Name:
Jakob
| Date:
16.08.2011
|
| Continue from Experiment: 12.08.2011
Order primers
|
| Project Name:
Red light receptor, amplify Cph8
|
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl
| H20
| Name
|
| 10µl
| 5x Phusion Buffer
| of Primer
|
| 2.5µl
| Primer fw
| P 58 (1:10)
|
| 2.5µl
| Primer dw
| P 59 (1:10)
|
| 1µl
| dNTPs
| of Template DNA
|
| 1µl
| DNA-Template
| JT122 5ng/µl
|
| 0.5 µl
| Phusion (add in the end)
|
|
The program we used: for Cph8
| Temp.
| Time
| Nr. of cycles
|
| 94°
| 5’
| 1x
|
| 94°
| 30’’
| 2x
|
| 68°
| 30’’
|
| 72°
| 2’30’’
|
| 94°
| 30’’
| 2x
|
| 67°
| 30’’
|
| 72°
| 2’30’’
|
| 94°
| 30’’
| 5x
|
| 66°
| 30’’
|
| 72°
| 2’30’’
|
| 94°
| 30’’
| 25x
|
| 65°
| 30’’
|
| 72°
| 2’30’’
|
| 72°
| 7’
| 1x
|
| 4°
| ∞
|
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
Ligation
Investigators: Sophie
Ligation of IPTG-promoter BBa_J04500, GFP-pbd and psb1c3-vector
Transformation
Investigators: Sophie
Transformation of ligation product
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