Team:Freiburg/Notebook/24 August

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green light receptor

Digest of CcaR

Investigators: Jakob

• 10µl BSA
  
• 10µl NEB
  
• 2µl DpnI
  
• 38µl DNA
  

Digest for 2h at 37°C

Transformation of CcaR

Investigators: Jakob

Testdigest of quickchanged CcaS

Investigators: Julia

today I prepared 9 minipreps of the the quickchanged CcaS.
They were digested with EcoRI and loaded on a gel.
Those which are not cut twice were changed during PCR correctly.
We will send them for sequencing today.

gel loaded with: Ladder 1 kb, qS50 a,b,c,d , qS51 a, b, c, d
lower lane:Ladder 1 kb qS51 e

positive clones are qS50b and qS51a.
concentration of minpreps: qS50b: 88,6 ng/μl and qS51a: 81,1 ng/μl.

blue light receptor

Transformation

Investigators: Sandra

Repeat of the transformation from yesterday with the Lovtap-NotGate-pSB1A3 vector.

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

20 cycles with an annealing temperature of 65/68/71 ºC (gradient-PCR) and touchdown then 10 cycles with an annealing temperature of 65/70/75 ºC and touchdown
digested with DpnI

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?
the PCR-products are labelled Not, nOt and noT and are stored in the "unverdaute Minipreps II"-box (or in the Gibson box?) If Sequencing is correct, I will do a new 3A assembly with the LovTAP-PCR product and an Amp-vector next.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

NAME OF YOUR EXPERIMENT

PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

What program do you use?

Annealing 60-70

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?