Team:Paris/August 8

Minipreps : Plasmid extraction

Extraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.

• Carried out 2 times (2 tubes)

Amplification of Genes of interest (OmpR, EnvZ, FlhDC)

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR Protocol

• List of Oligos :

Protocol PCR

For each samples,

• Preparation of the templates :
  --> Resuspend of 1 colony in 100µl of water.

• Preparation of PCR mix :

1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

• Program PCR "Screening": Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Electrophoresis Purification of PCR

When the PCR cycles were finished,

conditions :

• 10µl of ladder 1 kb (unlike 100 pb)
• 2 x 30µl of PCR products added with 10µl of loading Dye 6x
• migration ~30min at 100W on a 1,5% agarose gel.

Results of electrophoresis

gel 1 150px gel 2 145px

==> Conclusion:

• After electrophoresis, the bands corresponding to the right amplification were excised and purified using the Promega protocol.
• Elution in 30 µL of buffer EB.
• Store at -20°C

Transformation results

PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR