Team:Freiburg/Notebook/22 July

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Precipitator

Miniprep

Qiagen Kit

Documentation:

Why are you doing this experiment? Name the parts which you extract.

Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.

Testdigest

For one reaction you need: For Mastermix: Number of samples+2extra

DNA from todays Miniprep. See labeling there.

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!

Meeting

attendants: Jakob, Julia, Manuel, Ruediger, Sandra, Sophie, Theo (later), Tobi time: 9:00 - 10:30

green light receptor

already done:

• CcaS was amplified again from the genomic DNA

To-do:

• 3-A-Assembly of CcaR into an empty vector with a medium promotor
• 3-A-Assembly of CcaS into an empty vector with a weak promotor
• 3-A-Assembly of CcaR and CcaS to get the final construct

blue light receptor

already done:

• Primer-Design for the Gibson-Assembly
• growth medium without tryptophane is already ordered

To-do:

• amplify the NOT-Gate

red light receptor

already done:

• one positive colony from the 3-A of ho1 and the terminator
• no positive colony from the 3-A of pcyA and the terminator
• the PCR to amplify cph8 didn't give a product, we asked the team from Mexico to send us the cph8
• the team from Upsala (Sweden) has succeeded in amplifying the cph8 (according to their notebook)

To-do:

• 3-A-Assembly of the ho1-term with a mediumPromotor-mediumRBS

Lysis cassette

already done:

• the quick change didn't work the first time, we should repeat this
• possible alternative to express the lysis cassette could be: temperature regulated RNA

To-do:

• repeat the quick change

Precipitator

already done:

• the GFP-PBD was cloned (colonies showed up)

To-do:

• test digest and sequencing of the GFP-PBD (first have a look if you see the GFP fluorescence)
• possible test of the PBD: grow the E. coli, lyse them, put the lysate into a plate (96 well?), wash the wells to get rid of the other proteins, measure the GFP fluorescence with a plate reader
• microscope the E. coli cells

other suff

• think about possible give-aways for the sponsoring video
• jacob will create a Youtube account and upload the sponsoring video
• meeting for the wiki takeover: wednesday 9:00 am (new time: 12:15)
• modelling should be started now, either Ni-binding and release or GFP-PBD binding and release, Ruediger will do this

Lysis cassette

Quickchange PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?