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Team:Freiburg/Notebook/15 July
From 2011.igem.org
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Contents |
Meeting
attendants: Tobi, Theo, Julia, Rüdiger, Jakob, Sandra
blue light receptor
already done:
- Transformation of Lov-Tap in cells.
To-do:
- Cloning of promotor (medium) in Lov-Tap part and perform a transformation. Plating out of the cells on trypthophan-free medium.
Quick change
Investigators: Theo
already done:
- repressor part (BBa_K098995) has no bases between RBS (B0034) and start codon (K098997), resulting in no translation.
- quickchange of the repressor part to insert 6bp between RBS and start codon
To-do:
- DpnI digestion to digest the DNA template (methylated DNA) of the PCR to have only the new synthesized DNA strand.
- After digestion with DpnI, transformation of cells with the corrected part (BBa_K08995)
Lysis cassette
Digestion of Quickchange
| Name: Theo
| Date: 15.07.2011 |
| Continue from Experiment 14.07.2011
Quickchange PCR | |
| Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device | |
| 4,5μl | H2O | ||
| 4μl | Buffer, NEB4 | ||
| 4μl | BSA (10x) | ||
| 1,5 μl | Enzym 1 | DpnI | |
| 21 μl | DNA | Quickchange PCR |
35 μl total volume
Incubate for about 1h at 37°C + Heat inactivation at 80°C for 20min.
Transformation
| Name: Theo | Date: 15.07.2011 |
| Continue from 15.07.2011 Digestion of Quickchange
Experiment | |
| Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device | |
Procedure
- take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
- thaw cells on ice 20 minutes
- pipette 50 μl cells and 2 μl DNA into eppi still on ice!
- Incubate for 30 minutes on ice
- Heat at 42°C for 60 sec
- Incubate on ice for 5 minutes
- Add 200 μl LB Broth
- Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
- Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
Documentation:
Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
| It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.
|
Precipitator
PCR
| Name:
Ruediger | Date:
18.07.2011 |
| Continue from Experiment (Date)PCR 1507
(Name) Ruediger | |
| Project Name:
GFP Pbd | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | |
| 10µl | 5x Phusion Buffer | |
| 2.5µl | Primer fw | |
| 2.5µl | Primer dw | |
| 1µl | dNTPs | |
| 1µl | DNA-Template | |
| 0.5 µl | Phusion (add in the end) |
What program do you use?
10x (95C-41/52C-72C) + 25x ((95C-60C-72C)
How did you label the PCR-Product, where is it stored and what do you do next?
Reactions:
lane 1
quick load braod range marker
lane 2
empty
lane 3
P28+P18+M14.1
lane 4
P28+P19+M14.1
lane 5
P28+P20+M14.1
Results:
did not work well.
Strange band size in lane 3. 4 and 5 did not work.
