Team:Freiburg/Notebook/12 August

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Meeting

attendants: Jakob, Julia, Manuel, Sandra, Sophie, Theo, Tobias
Time: 10:00 - 11:30

green light receptor

already done:

• quick change with CcaR didn't work
• colonies for pcyA and ho1

To-do:

• order new primer for the quick change
• cph8: we will use the primer and protocol from the Upsalla team
• test if the colonies are positive

blue light receptor

already done:

• PCR to amplifiy the receptor with new primer pair

To-do:

• check if the PCR worked

red light receptor

already done:

To-do:

Lysis cassette

already done:

• lysis cassette with RBS
• colonies for the GFP-PBD
• inducible promotor + PR done

To-do:

• overnight culture of the GFP-PDB colonies, miniprep, test digest and sequencing

Precipitator

already done:

• waiting for the genes

To-do: still more waiting to do

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

Gel of the PCR product: LovTAP

Investigators: Sandra

To confirm the PCR worked this time, we loaded the sample onto a gel. The size of the fragment should be 1000bp. The gel showed a band at 1000bp. We digested the PCR product with DpnI and then purified the DNA with the PCR purification kit to further use the PCR product for 3A-assembly.

3A-assembly of Not-Gate, LovTAP in pSB1T3

Investigators: Sandra

Digestion

Amount of DNA and H20:

Enzymes necessary for digestion:

• Incubation at 37°C for 6 hours
• 20 minutes at 80°C

red light receptor

Picking clones of pcyA and ho1

Investigators: Sandra

Picked 2 clones of each plate:

• 1pTd (PR1+pcyA+terminator)
• 2pTd (PR2+pcyA+terminator)
• 1pTb (PR1+pcyA+terminator)
• 2pTb (PR2+pcyA+terminator)
• 1L23 (PR1+ho1+terminator)
• 2L23 (PR2+ho1+terminator)

Incubation over night.

Lysis cassette

Miniprep

Investigators: Jakob, Ruediger, Theo

Documentation:

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

• Need the DNA concentration

Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results. How did you label your probes and where are they stored?

Precipitator

Cloning

Investigators: Sophie
continue from experiment: Miniprep (Jakob/Ruediger, 12.8., see lysis cassette)
project name: inducible promoter for Pbd
parts: S54 (Eco & Spe)
GFP-Pbd ε5 (Xba 7 Pst)
psb1t3 (Eco & Pst)
stored in: Ruediger's box

Ligation

Investigators: Sophie
continue from experiment: Cloning (today, me)
project name: inducible promoter for Pbd
parts: S54 (Eco & Spe)
GFP-Pbd ε5 (Xba 7 Pst)
psb1t3 (Eco & Pst)
name of ligation product: IPTG+Pbd
stored in: Ruediger's box

Testdigest

Investigators: Sophie
continue from experiment: Ligation (today, me)
project name: inducible promoter for Pbd
PR6 6 and Pr 4 4 were sequenced but PRs were missing again. Inducible promoter seems to be necessary.