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Team:Freiburg/Notebook/5 August
From 2011.igem.org
Contact 
Contents |
Meeting
attendants: Jakob, Julia, Manuel, Rüdiger, Sandra, Theo, Tobi
time: 9:30 - 11:00
green light receptor
already done:
- CcaS: first quick change PCR removed the SpeI restriction site, EcoRI didn't work
- CcaR: first quick change PCR didn't work (EcoRI restriction site not removed), wrong PCR program
To-do:
- do the second quick change with the changed Ccas to remove the EcoRI restriction site
- repeat the quick change with the CcaR
- after the quick changes do a Gibson assembly with pcyA and ho1
- in parallel do 3A-assemblies to create the same construct as a backup plan
blue light receptor
already done:
- ordered new primers (the third pair)
- first primer pair had the overhang, second without overhang, third primer pair now without overhang and position shifted
To-do:
- try the gibson assembly again with the third primer pair
red light receptor
already done:
- 3A of pcyA and terminator sent for sequencing
- cph8 was sent for sequencing
- julia transformed the ompR promotor
To-do:
- add a Promotor-RBS to the pcyA-Terminator
- if the sequence is OK, amplify the part with the overhangs
- add terminator and Promotor-RBS
- do a miniprep of the ompR promotor
Lysis cassette
already done:
- 3A-assembly of the lysis casette
- 3A-assembly oth the GFP-PBD
- quick change PCR of the lysis casette (11 bases to insert the RBS)
To-do:
- do a miniprep from all three attempts
Precipitator
already done:
- waiting for godot
To-do:
- waiting for godot
other stuff
- start creating nice pictures to explain your subproject and write text to explain the pictures
- Sandra organized Freehand from the MPI, it is installed on both mac books
- give-aways: digital postcard, real postcard, fluorescent protein
- Tobi will ask at the financial department about the croud funding money
- Rüdiger will get us more media attention
- cooperation with other german teams: ask munich to do an appointment for a skype conforence about the light inducible promotors
- cooperation with Upsala: find an appointment for the skype talk
green light receptor
1.Quickchange
Investigators: Jakob
PCR
| Name:
Jakob | Date:
05.08.2011 |
| Continue from Experiment: 05.08.2011
Quickchange of Spe1 (Sequencing was correct) | |
| Project Name:
Green light receptor | |
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 | P14 (1:10) |
| 2.5µl | Primer dw | P15 (1:10) |
| 1µl | dNTPs | of Template DNA |
| 1µl | DNA-Template | CcaS (120ng/µl) and CcaS (130ng/µl) dilute to 5ng/µl |
| 0.5 µl | Phusion (add in the end) |
What program do you use?
| 95 °C – 5’ | 95°C – 15 ‘’ | x 20 | 72°C – 5’ | 4°C – ∞ |
| 55°C – 15 ‘’ | ||||
| 72°C – 1,15’ + 2’’/Cycle |
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
How did you label the PCR-Product, where is it stored and what do you do next?
Labeled as Jakob1 and Jakob2 in the fridge
2.Digest quickchange PCR with Dpn1
5µl Buffer NEB4
5µl BSA (10x)
2µl Dpn1
38µl DNA from quickchange (CcaS)
incubate at 37°C for 1,5 h
inactivate at 80°C for 20 min.
3 . T r a n s f o r m a t i o n
4.Picked some colonies
from transformation yesterday
- To-do: pick colonies, do Miniprep
blue light receptor
Gradient PCR of Lovtap with new primer
Investigators: Sandra
Primer:
- P38 (former name:P36) LOVdw
- P42 LOVtap up
DNA template:
- M35 (Lovtap)
PCR program:
- "Lovtap ohne Überhänge" with temperature gradient during the annealing step from 50°C-60°C. The elongation step and the final elongation step were extended.
"cold":50°C
"warm":60°C
Comments: PCR did not work again. We will try to do 3A-assembly.
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
