Team:Freiburg/Notebook/5 August

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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

PCR-Mixture for one Reaction:

For a 50 µl reaction use

Program I used

• 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.

• To-do: Transformation

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

Lysis Cassette +RBS

Investigators:Theo

No colonies, repeated transformation as August 4th

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME