Team:Freiburg/Notebook/28 July

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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.

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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.

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

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.

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NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME