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Team:Freiburg/Notebook/22 July
From 2011.igem.org
Contact 
Contents |
Precipitator
Miniprep
Qiagen Kit
| Name:
Sophie | Date:
22.07 |
| Continue from Experiment (Date) Trafo 20.07 Ruediger
(Name) | |
| Project Name:
GFPpbd | |
Documentation:
Why are you doing this experiment? Name the parts which you extract.
| 1abc: S39+M14+P28+P18
2abc: S39+M14+P28+P19 3abc: S39+M14+P28+P20 4abc: S43+M14+P28+P18 5abc: S43+M14+P28+P19 6abc: S43+M14+P28+P20
|
Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.
| Mistakes: some lids broke
nanodrop labeling partly confused |
| Sample ID | Nucleic Acid Conc. | Unit | 260/280 | 260/230 |
| 1a | 60.6
| ng/µl | 1.85
| 1.75
|
| 1b | 102.8
| ng/µl | 1.66
| 0.96
|
| 1c | 41.5
| ng/µl | 1.74
| 1.63
|
| 1d | 85.5
| ng/µl | 1.58
| 1.09
|
| 2a | 72.4
| ng/µl | 1.62
| 1.04
|
| 2b | 9.7
| ng/µl | 0.86
| 0.34
|
| 2c | 116.8
| ng/µl | 1.64
| 0.9
|
| 2d | 38.5
| ng/µl | 1.56
| 0.95
|
| 3a | 86.5
| ng/µl | 1.56
| 0.75
|
| 3b | 77.9
| ng/µl | 1.57
| 0.89
|
| 3c | 94
| ng/µl | 1.66
| 1.07
|
| 3d | 88.1
| ng/µl | 1.64
| 0.95
|
| 4a | 91.3
| ng/µl | 1.64
| 0.91
|
| 4b | 93.4
| ng/µl | 1.67
| 1.09
|
| 4c | 111.5
| ng/µl | 1.7
| 1.15
|
| 4d | 71.5
| ng/µl | 1.86
| 1.95
|
| 5a | 73.2
| ng/µl | 1.81
| 1.75
|
| 5b | 172.3
| ng/µl | 1.79
| 2.01
|
| 5c | 53
| ng/µl | 1.61
| 1.98
|
| 5d | 97
| ng/µl | 1.72
| 1.43
|
| 6a | 92
| ng/µl | 1.7
| 1.27
|
| 6b | 92.8
| ng/µl | 1.62
| 1.05
|
| 6c | 81.3
| ng/µl | 1.66
| 1.14
|
| 6d | 106.8
| ng/µl | 1.73
| 1.26
|
Testdigest
| Name:
Ruediger | Date:
21.07 |
| Continue from Experiment (Date)
Miniprep Sophie (Name) | |
| Project Name: GFPPbd | |
For one reaction you need: For Mastermix: Number of samples+2extra
| 4μl | H2O | 104 | |
| 1μl | Buffer, NEB4 | 26 | |
| 1μl | BSA (10x) | 26 | |
| 0,5 μl | Enzym 1 | ||
| 0,5 μl | Enzym 2 | ||
| 3 μl | DNA |
DNA from todays Miniprep. See labeling there.
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.
Take a picture of the gel, print picture and label the lanes!
Meeting
attendants: Jakob, Julia, Manuel, Ruediger, Sandra, Sophie, Theo (later), Tobi time: 9:00 - 10:30
green light receptor
already done:
- CcaS was amplified again from the genomic DNA
To-do:
- 3-A-Assembly of CcaR into an empty vector with a medium promotor
- 3-A-Assembly of CcaS into an empty vector with a weak promotor
- 3-A-Assembly of CcaR and CcaS to get the final construct
blue light receptor
already done:
- Primer-Design for the Gibson-Assembly
- growth medium without tryptophane is already ordered
To-do:
- amplify the NOT-Gate
red light receptor
already done:
- one positive colony from the 3-A of ho1 and the terminator
- no positive colony from the 3-A of pcyA and the terminator
- the PCR to amplify cph8 didn't give a product, we asked the team from Mexico to send us the cph8
- the team from Upsala (Sweden) has succeeded in amplifying the cph8 (according to their notebook)
To-do:
- 3-A-Assembly of the ho1-term with a mediumPromotor-mediumRBS
Lysis cassette
already done:
- the quick change didn't work the first time, we should repeat this
- possible alternative to express the lysis cassette could be: temperature regulated RNA
To-do:
- repeat the quick change
Precipitator
already done:
- the GFP-PBD was cloned (colonies showed up)
To-do:
- test digest and sequencing of the GFP-PBD (first have a look if you see the GFP fluorescence)
- possible test of the PBD: grow the E. coli, lyse them, put the lysate into a plate (96 well?), wash the wells to get rid of the other proteins, measure the GFP fluorescence with a plate reader
- microscope the E. coli cells
other suff
- think about possible give-aways for the sponsoring video
- jacob will create a Youtube account and upload the sponsoring video
- meeting for the wiki takeover: wednesday 9:00 am (new time: 12:15)
- modelling should be started now, either Ni-binding and release or GFP-PBD binding and release, Ruediger will do this
Lysis cassette
Quickchange PCR
| Name: Theo
| Date: 22.07.2011 |
| Project Name: Quickchange V.3 with correct lysis template | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | Name |
| 10µl | 6x Phusion HF Buffer | |
| 2.5µl | Primer fw | P29 (1:10) |
| 2.5µl | Primer dw | P30 (1:10) |
| 1µl | dNTPs | |
| 1µl | DNA-Template | S11 (->diluted to 10ng/µl) |
| 0.5 µl | Phusion (add in the end) |
What program do you use?
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