Week 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

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

Data:

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

For PCR we chose to use … ng of primer (V=…µL). As we know the size of our primers, we could define the number of primers that we have to use to begin the reaction. We define the number of recquired matrix DNA in terms of concentration of sample with the following formula:

AJOUTER LA FORMULE

Analysis of Agarose gel's results:

AJOUTER LA PHOTO LEGENDEE

Extraction of natural promoter sequence

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

We remake PCR samples that failed the previous week.

• Fec A
• Ent C
• Fec C
• Ace B

PCR products are placed on gel and purificate. After, PCR products are tested with 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

Tris-HCl solution preparation(1M) : Stock solution 100X

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

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

1. Disolve 4,6g of Trisbase in 30 mL of distilled water.
2. Adjust pH with concentrated HCl(~4 mL) until pH=7,5.
3. Add distilled water until 50 mL then put in autoclave.

Note: If the solution has a yellow coloration, do the preparation again with better Trisbase.

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.

Kanamycin preparation

Preparation of 3 tubes of 1.5 mL of Kanamycin.

Thursday, July 11

Preparation of primers solution:

1. Centrifugate 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 Cyrille's samples:

• Terminator (T)
• Promotor (P)
• Plasmid 1K3
• Plasmid 1C3
• sfGFP

These transformations allow us to do glycerols of these constrctions.

PCR on E.coli genom (BG1655 strain)

• 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

Transformations test

• Terminator (T) = OK
• Promotor (P) = OK
• Plasmid 1K3 = OK
• Plasmid 1C3 = OK
• sfGFP = OK

Note : The negative control was suspect.

Petri dishes are le on the bench at room temperature during the week-end, colonies will be reisolate next week.

Migration of samples of PCR extraction.

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