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4: 8^th July - 14^th July

Monday, July 8th

We prepared the BL21 and BG1655 strains to be competent.

Tuesday, July 9th

Both strain are not contaminated.

BL21 are highly competent, BG1655 are little competent.

Wednesday, July 10th

PCR procedure optimization

But : Aim: Define the number of matrix of optimal DNA to amplify a gene by PCR.

Information:

• [primer] = --- ng/µl
  
• size(primer) = --- nt
  

For PCR we chose to use … ng of primer (V=…µL). Same we know size of our primer, we could define the number of primer that we use begin the reaction. Thanks this expression we define the number of template to take in terms of concentration of sample.

AJOUTER LA FORMULE

Analysis of results of agarose gel:

AJOUTER LA PHOTO LEGENDEE

Extraction of sequence of wild promoter.

Aim: we want have sequences of promoters of genes under the control of transcription factor FUR (Ferric Uptake Regulation).

We remake PCR samples that failed the last week.

• Fec A
• Ent C
• Fec C
• Ace B

PCR products are placed on gel and purifiate. After, PCR products are tested in nanodrop.

Fec A (BG1655) [c] = 23.7 ng/µl 260/280 = 1.90

Fec A (BL21) [c] = 53.1 ng/µl 260/280 = 1.73

Ent C (BG1655) [c] = 52.4 ng/µl 260/280 = 1.71

Ent C (BL21) [c] = 50.0 ng/µl 260/280 = 1.94

Ace B (BG1655) [c] = 46.5 ng/µl 260/280 = 1.89

Fec C (BG1655) [c] = 34.2 ng/µl 260/280 = 1.91

Training a tris-HCl solution (1M) : First solution 100X

Aim: Tris-HCl solution will use in a final concentration of 10 mM to resuspend our primers.

Preparation of the stock solution (1 M, pH 7.5) to a volume of 50 mL:

1. Dissolve 4.6g of trisbase in 30 mL of water.
2. Adjust pH of this solution at 7.5 with concentrated HCl ([c] = --- mol/L // volume ajouter ~ 4 mL).
3. Adjust volume of the solution at 50 mL with water.
4. Autoclave the solution.

Note: If the solution is yellow, we should repeat with Tris of best quality.

Preparation of the solution (10mM, pH 7.5) for a volume of 50 mL.
Take 50µL of the stock solution at 1M and add 49.5 mL of water for the dilution.

Préparation of Kanamycine

Preparation of 3 solution of 1.5 mL of Kanamycine.

Thursday, July 11

Preparation of solution of primer:

1. Centrifuge tubes at 8000 rpm, begin 30 seconds.
2. Add 250 µL of Tris HCl (10 mM); [Primer] = 100µM

We prepare a diluted solution at 5 µM from to the stock solution, for PCR reactions.
Dilution at 1/20: We take 5 µL of the stock solution (100 µM) and we diluate in 95 µL of tris-HCl (10 mM).

Transformation of BL21:

Chemical transformation of BL21 with samples of Cyrille:

• Terminateur (T)
• Promoteur (P)
• Plasmide 1K3
• Plasmide 1C3
• sfGFP

These transformations allow us to do glycerols of these constrctions.

PCR sur le génome de E.coli (souche BG1655)

• Fep A (Primers P021 and P022)
• Fes (Primers P023 and P024)
• sdh C (Primers P025 and P026)
• ybi L (Primers P027 and P028)
• ync E (Primers P029 and P030)

Friday, July 12

Vérification of transformations

• Terminateur (T) = OK
• Promoteur (P) = OK
• Plasmide 1K3 = OK
• Plasmide 1C3 = OK
• sfGFP = OK

note : Note: The negative control was suspect.

Les boites de Pétri sont laissés sur la paillasse à température ambiante pour le week-end, les colonies seront repiquée la semaine prochaine.

Migration of samples of PCR extraction.

The promoter sequence of genes Fep A, Fes, Sdh C, ybi L and ync E were extracted with successfully.
Samples were purified and, after, tested by nanodrop.

Fep A (BG1655) [c] = 38.3 ng/µl 260/280 = 1.93

Fes (BG1655) [c] = 45.5 ng/µl 260/280 = 1.89

Sdh C (BG1655) [c] = 28.5 ng/µl 260/280 = 1.86

ybi L (BG1655) [c] = 31.0 ng/µl 260/280 = 2.09

ync E (BG1655) [c] = 25.1 ng/µl 260/280 = 1.93

Voir s'il ne faut pas faire le tableau recap des extractions

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