Team:Freiburg/Notebook/28 July

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green light receptor

Digest of Qickchange PCR product with DpnI

Investigators:Julia
in order to get rid of old plasmids a digest with DpnI was performed.

Transformation

Competent cells (50µl) were transformed with 10µl of the PCR- Product after iGEM Protocol.
CcaS is in the iGEM vector pSB1K3
CcaR in pSB1C3.

blue light receptor

Digestion of PCR product

Investigators:Sandra

Digest of PCR product S35 (Lov-tap), because PCR did not work out. We got a band of 1000bp, but we expected 3000bp.
Digest:

• 1 μl NEB4 buffer
  
• 1 μl BSA
  
• 1 μl NcoI
  
• 2 μl H2O
  
• 5 μl S35
  

Incubation for 30 min 37°C.
Afterwards the digested product was loaded onto gel.
Comments: Not expected results. We will try a gradient PCR.

Gradient PCR for Lov-tap S35

Investigators: Sophie

Comments: Because PCR for S35 did not work, we are doing a gradient PCR.
PCR as usual. Only temperature were adjusted, to determine the optimum of the annealing temperature.

• 54-59°C
• 64-72°C

After the PCR we will load the samples onto a gel to see the results of the gradient PCR.
PCR did not work, only bands with 1000bp instead of 3000bp. We will order new Primers and then try again.

Miniprep of Not-Gate

Investigators: Sophie

Picked colonies from plate and then performed a miniprep.

DNA-concentration measured with nanodrop:

Testdigest of Miniprep

Investigators: Sophie

Testdigest of M45a-M45d.

• 4 μl H20
  
• 1 μl NEB4
  
• 1 μl BSA
  
• 0.5 μl EcoRI
  
• 0.5 μl PStI
  
• 3 μl DNA (M45a-M45d)
  

Incubation for 1 hour. Gel was loaded afterwards.
Comments: M45c and M45d look good.

red light receptor

Miniprep of BBa_I15008 and BBa_I15009

Investigators:Julia

because there was some confusion with the labbeling and no stock, I am repeating transformation and DNA extraction.

Precipitator

Miniprep

Investigators: Rüdiger

Miniprep with Hiss-kit. Samples from yesterday were inoculated:

• 43-20 a-d
• 43-19 a-d
• 43-18 a,b
• 39-19
• 39-20