Team:Paris Saclay/Notebook/August/25

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(Difference between revisions)
(Electrophoresis of the digestion product of pGEMTeasy+chromoprotein)
(B - Construction of the fusion protein)
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We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction
We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction
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===D - Lemon Scent===
===D - Lemon Scent===

Revision as of 09:08, 26 August 2014

Contents

Monday 25th August

Lab work

B - Construction of the fusion protein

plasmid extraction : pGEMTeasy + chromoprotein

by Laetitia

We used the bacteria containing pGEMTeasy + chromoprotein launched by Melanie sunday the 24th for the plasmid extraction, 6 independent cultures which come from 6 independent colonies.

We made a stock of bacteria for each sample (x6).

To extract the plasmid, we used the plasmid DNA purification kit (Macherey-Nagel).

Digestion of pGEMTeasy+chromoproteinby XbaI and PstI

by Laetitia

The goal is to check the presence of the insert inside the plasmid. We digest the 6 samples of purified plasmid.

component volume
Plasmid 5 μl
Fast Digest buffer 10X 1μl
XbaI 0.5μl
PstI 0.5μl
H20 3μl

1h at 37°C

Electrophoresis of the digestion product of pGEMTeasy+chromoprotein

by Laetitia

T : control negatif without enzyme

D : sample digested by XbaI and PstI

T1 - D1 - T2 - D2 - T3 - D3 - E - T4 - D4 - T5 - o - D6

250814 laetitia digestion verif chromoP pGEMTeasy.jpg

Results : lost of the sample D5 in a hole in the agarose gel

The samples 3, 4 and 6 seems to contain the chromoprotein gene (two bands)

--> We will try another plasmid extraction because the electrophoresis profil doesn't show bands for every sample.

Bacterial culture of DH5a containing pGEMTeasy + chromprotein

by Laetitia

We launched 6 cultures in 5mL LB + Ampi (1/1000) - 37°C at night . The bacteria come from the stock made before the plasmid extraction

D - Lemon Scent

Gel electrophoresis of CAD

by Sean

Legend

  1. ladder 10µl
  2. PCR result of CAD

Paris Saclay Sean 140825.jpg

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