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Team:Freiburg/Notebook/5 August
From 2011.igem.org
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==<span style="color:green;">green light receptor</span>== | ==<span style="color:green;">green light receptor</span>== | ||
| - | ===Quickchange=== | + | ===1.Quickchange=== |
'''Investigators: Jakob''' | '''Investigators: Jakob''' | ||
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Labeled as Jakob1 and Jakob2 in the fridge | Labeled as Jakob1 and Jakob2 in the fridge | ||
| - | ===Digest quickchange PCR with Dpn1=== | + | ===2.Digest quickchange PCR with Dpn1=== |
'''Investigator: Jakob''' | '''Investigator: Jakob''' | ||
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5µl BSA (10x) <br/> | 5µl BSA (10x) <br/> | ||
2µl Dpn1 <br/> | 2µl Dpn1 <br/> | ||
| - | 38µl DNA from quickchange ( | + | 38µl DNA from quickchange (CcaS) <br/> |
incubate at 37°C for 1,5 h <br/> | incubate at 37°C for 1,5 h <br/> | ||
inactivate at 80°C for 20 min. <br/> | inactivate at 80°C for 20 min. <br/> | ||
<br/> | <br/> | ||
| + | |||
| + | ===3.Picked some colonies=== | ||
==<span style="color:blue;">blue light receptor</span>== | ==<span style="color:blue;">blue light receptor</span>== | ||
Revision as of 14:26, 5 August 2011
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
Investigator: Jakob
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.Picked some colonies
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
