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 amplification

Protocol

• List of Oligos :

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

• Preparation of PCR mix :

For each samples,

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

PCR verification/Analyse

After the PCR :

• 3µl have been analysed by electrophoresis
• the other 47µl of PCR products have been purified by the Promega kit.

Electrophoresis

• ladder : 10µl ladder 1 kb
• samples : 3µl of PCR products + 2µl of Loading Dye
• Conditions : migration 30min at 100W, on a 1% agarose gel

• Results :

Quantification of the PCR products purified

• Blank : 2µl of buffer EB + 98µl of water
• Samples : 2µl of PCR purified + 98µl of water.

• Results :

Digestion of PCR products

Protocol :

• Incubate 2h30 at 37°C

Purification/Analysis

Electrophoresis

• ladder : 10µl ladder 1 kb (for 1%) or ladder 100pb ( for 1.5%)
• samples : 3µl of insert + 2µl of Loading Dye
• Conditions : migration 30min at 100W, on a 1% agarose gel for the vector and 1.5% for the insert.

• Results :

Transformation results

PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR

Protocol of screening PCR

• Mix

• 50µl of Mix PCR by tube/clone
• one toothpick of each clone's colony by tube
• Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

• 10µl of ladder 100 pb
• 10µl of screening PCR
• migration ~30min at 100W on 1,5% gel

• 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 gel 2

==> Conclusion:

• PCR of pFlgA, pFlgB and pFlhB have succeed, but we always have a problem with pFlhDC probably because of the oligos wich are not specific.