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Team:Freiburg/Notebook/29 August
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the lane labeled P, is the PCR product.Expected size: 264 bp | the lane labeled P, is the PCR product.Expected size: 264 bp | ||
| - | ===Testdigest of quickchanged CcaR=== | + | ===Testdigest of quickchanged CcaR and Precipitator=== |
'''Investigators:Julia,with help of the team''' | '''Investigators:Julia,with help of the team''' | ||
| + | <br/> | ||
| + | <br/><br/> | ||
| + | 3μl of the Miniprep were digested with EcoRI and PstI.<br/> | ||
| + | [[File:Freiburg11-08-29 Testdigest.jpeg|400px|400px]] | ||
| + | <br/><br/><br/> | ||
| + | we are going to sequence the Precipitator samples 61a and b<br/> (wrong labeling on the pic, its the 63 in the right corner)<br/>62 b and f<br/>63 c and d <br/>and of the green light response regulator qCcaR 17b and 18a<br/> | ||
==<span style="color:blue;">blue light receptor</span>== | ==<span style="color:blue;">blue light receptor</span>== | ||
Revision as of 13:28, 30 August 2011
Contact 
Contents |
green light receptor
PCR to amplify Green light Promotor PcpcG
Investigators:Julia PCR
| Name: Julia
| Date: 29.08.2011 |
| Project Name: Amplification of Green light Promotor | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | |
| 10µl | 5x Phusion Buffer | |
| 2.5µl | Primer fw | tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC |
| 2.5µl | Primer dw | tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC |
| 1µl | dNTPs | |
| 1µl | DNA-Template | original Synechocystis genome |
| 0.5 µl | Phusion |
What program do you use?
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
the lane labeled P, is the PCR product.Expected size: 264 bp
Testdigest of quickchanged CcaR and Precipitator
Investigators:Julia,with help of the team
3μl of the Miniprep were digested with EcoRI and PstI.
we are going to sequence the Precipitator samples 61a and b
(wrong labeling on the pic, its the 63 in the right corner)
62 b and f
63 c and d
and of the green light response regulator qCcaR 17b and 18a
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
- pSB1C3
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: new
| |
| 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
| 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 (Bba_Q04400) |
| 0.5 µl | Phusion (add in the end) |
The program we used: RT 2step (see last PCR of NOT-gate)
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
