Team:Freiburg/Notebook/22 July

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Precipitator

Miniprep

Qiagen Kit

Procedure:

1.Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm

(6800 x g) for 3 min at room temperature (15–25°C).

2.Resuspend pelleted bacterial cells in 250 ␣l Buffer P1 and transfer to a microcentrifuge tube.

3.Add 250 ␣l Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.

4.Add 350 ␣l Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.

5.Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

6.Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting.␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.

7.Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB. ␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.

Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.

8.Wash the QIAprep spin column by adding 0.75 ml Buffer PE. ␣Centrifuge for 30–60 s and discard the flow-through, or␣apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source. Transfer the QIAprep spin column to the collection tube.

9.Centrifuge for 1 min to remove residual wash buffer.

10.Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 ␣l Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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