Team:Paris/July 25

Extraction of the DNA

• Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
• Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))

• Test of the succeed of the extraction by electrophoresis on 2µl of the parts extracted.

Determination of DNA concentration by spectrophotometry

conditions: 2µl of the DNA extracted 98µl of pure water

• Reading against the adaptated blank (2µl of the elution's buffer / 98µl of pure water)

• List of the results :

D100 --> D110 cl-1 ES : Absorbance ~20-50µg/ml ; DO 260/280 = 1,5 ; DO 260/230 = 0,0-0.20 D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml

Determination of DNA concentration by electrophoresis

• to check for the samples from Qiagen's protocol, if the absence of detection of absorbance, is due to a problem of reading or a problem during the extraction, we realise an electrophoresis.

==> conclusion : we don't succeed to detect DNA by spectrophometry or electrophoresis for the samples produced by Qiagen's protocol.

==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments

Culture

MiniPreps

• Use of Promega's protocol on all the clones cultivated on the 24th.
• Preparation of 50µl of minipreps in simple.

list of Minipreps