Meeting
attendants: Julia, Manuel, Rüdiger, Sandra, Theo, Tobi
Time: 9:00 - 11:00
green light receptor
already done:
- Ccas has to sites where we need to do a mutagenesis (quick change), both were done seperately, but the no colonies showed up (wrong PCR program)
- CcaR has one site where we need to do a mutagenesis (quick change), this has been done, colonies were growing
To-do:
- miniprep, digestion and sequencing of the CcaR clones
- do the Ccas PCR again
- next steps would be the big assembly wiht pcyA
blue light receptor
already done:
- PCR of Lov-tap and Not-gate. Not-gate sequencing was good. PCR of Lov-tap did not work the first time and the second time with the temperature gradient PCR
To-do:
- creating new primer for the Lov-tap as the old ones had a high probabylity for secondary structures
red light receptor
already done:
- waiting for cph8 from Mexico
- for pcyA we did a new transformation from the original plasmid
To-do:
- over night culture, miniprep and stock from the pcyA plates
- 3-A-Assembly of pcyA and Terminator
Lysis cassette
already done:
- the quick change didn't work the first time (wrong template DNA)
- second time went well, but the transformation was difficult (only 10 colonies)
To-do:
- do the miniprep and the sequencing
Precipitator
already done:
- GFP-PDB: cloning doesn't work only a few colonies
To-do:
- ask again if the gene synthesis is done: Rüdiger
- Gibson cloning with the GST-Tag, TEV-site and Promotor-RBS
other stuff
- deadline for the parts: 10th of september
- name all the files you upload on the wiki with the prefix Freiburg2011_
- start documenting the lab stuff in the wiki
- still missing: logo and text from hiss, MPI, Serva, Invitrogen
- Modelling: Rüdiger will ask Simon if he can help us along
- sponsoring give-aways: key tag, postcards, movie, pencils
- Lab tracks: Tobi wants to try it
- cooperation with Upsala: Yeahhh!
blue light receptor
Glycerol stocks of Not-Gate
Investigators: Sandra
M45d was sequenced and we did glycerol stocks of bacteria containing M45d.
Lysis cassette
Sequencing of Lysis Cassette V.2 (Quickchange-modified K098995 + Lysis genes K124017)
Test Sequencing showed that the parts were assembled correctly.
File #1: You can see the EcoRI, XbaI sites and also the Quickchange-modified K098995 as well as the XbaI-SpeI SCAR and the start of the Lysis genes K124017
File #2: You can see part of the Lysis genes K124017 and the SpeI and PstI sites
Right-click to download the annotated ape (.gb) file #1
Right-click to download the annotated ape (.gb) file #2
What can immediately be noticed is the absence of a RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017.
This had skipped our attention at the planing phase and was a source of headaches during the next days... :)
Precipitator
Testdigest
| Name:
Ruediger
| Date: 29.07
|
| Continue from Experiment (Date) Miniprep 28.07
(Name)
|
| Project Name: GFP Pbd
|
For one reaction you need: For Mastermix: 12 Number of samples+2extra
| 4μl
| H2O
| 84
|
|
| 1μl
| Buffer, NEB4
| 14
|
|
| 1μl
| BSA (10x)
| 14
|
|
| 0,5 μl
| Enzym 1
| 7
|
|
| 0,5 μl
| Enzym 2
| 7
|
|
| 3 μl
| DNA
| 1
|
|
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!
nothing worked, no GFP inserts seens anywhere. Gel picture was not saved by machine.
-Sequencing: no GFP in constructs - retry by Theo.
"
Contact 
