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Team:Freiburg/Notebook/3 August
From 2011.igem.org
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Add 1 μl Loading dye buffer and load the gel. | Add 1 μl Loading dye buffer and load the gel. | ||
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Revision as of 17:39, 18 September 2011
Contact 
Contents |
green light receptor
Testdigest
Investigators:Jakob, Julia
| Name:
Jakob | Date:
02.08.2011 |
| Continue from Experiment 02.08.2011
Miniprep | |
| Project Name: green light receptor, testdigest of CcaR with BamHI | |
For one reaction you need: For Mastermix: Number of samples+2extra
| 4μl | H2O | 20 | |
| 1μl | Buffer, NEB4 | 5 | |
| 1μl | BSA (10x) | 5 | |
| 0,5 μl | Enzym 1 | 1 | BamHI |
| 0,5 μl | Enzym 2 | - | |
| 3 μl | DNA | 3 |
10 μl total volume
| 4μl | H2O | 20 | |
| 1μl | Buffer, NEB4 | 5 | |
| 1μl | BSA (10x) | 5 | |
| 0,5 μl | Enzym 1 | 1 | EcoRI |
| 0,5 μl | Enzym 2 | 1 | PstI |
| 3 μl | DNA | 3 |
10 μl total volume
Give 3 μl of DNA in an eppi and add 7μl of the mastermix.
Incubate for about 1h at 37°C.
Add 1 μl Loading dye buffer and load the gel.
- Picture of the gel
CcaR digested with BamH1 are negativ
- To-do: quickchange (scroll down)
Quickchange
Investigator:Jakob
PCR
| Name:
Jakob | Date:
03.08.2011 |
| Continue from Experiment: | |
| Project Name:
Quickchange CcaR | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | |
| 10µl | 5x Phusion Buffer | |
| 2.5µl | Primer fw | Primer: P12 |
| 2.5µl | Primer dw | Primer: P13 |
| 1µl | dNTPs | |
| 1µl | DNA-Template | DNA: CcaR |
| 0.5 µl | Phusion (add in the end) |
results of transformation from 2nd Aug
Investigators:Julia
transformation worked!
blue light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
red light receptor
Colony picking
Investigator:Jakob
Picked some colonies for grow over night
- to-do: Miniprep
Lysis cassette
Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG
Primers for Quickchange
- Forward Primer: GGCGGTGATAATGGTTGCTAaagaggagaaaCTAGAGATGCCAGAAAAAACATGAC
- Reverse Primer: GTCATGTTTTTTCTGGCATCTCTAGtttctcctcttTAGCAACCATTATCACCGCC
Quickchange PCR
| Name: Theo
| Date: 03.08.2011 |
| Project Name: Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | Name |
| 10µl | 6x Phusion HF Buffer | |
| 2.5µl | Primer fw | P55 (RBS2LysV2up) (1:10) |
| 2.5µl | Primer dw | P56 (RBS2LysV2dw) (1:10) |
| 1µl | dNTPs | |
| 1µl | DNA-Template | S49 (Composite part -modified K098995+K124017) (->diluted to 5ng/µl) |
| 0.5 µl | Phusion (add in the end) |
What program do you use?
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Digestion of Quickchange with DpnI
| Name: Theo
| Date: 03.08.2011 |
| Continue from Experiment 03.08.2011
Ready Lysis Cassette (Part S49) + RBS | |
| Project Name: Ready Lysis Cassette (Part K124017) + RBS (B0034) | |
| 5μl | Buffer, NEB4 | ||
| 5μl | BSA (10x) | ||
| 2,5 μl | Enzym 1 | DpnI | |
| 37,5 μl | DNA | Quickchange PCR |
50 μl total volume
Incubate for about 1,5h at 37°C + Heat inactivation at 80°C for 20min.
Precipitator
Investigators: Theo
new 3A-assembly with Kan vector to see if pSB1T3 played a negative role in the last 3A-assembly.
Incubation over night at 16°C.
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
