Team:Paris/19 August 2009

NoteBook
August 19th

 

Lab work
Sequencing w/ Bobode

5 clones pLac/RBS-TetR have to be sequenced

Clone 1, 2, 6, 7, 8 used (because of their high DNA concentration)

Transformation w/ Bobode

pSB1A3-TolRII ligation was transformed into P10 chemical competent cells, using the "heat shock" protocol

Glycerol stock w/ Charlie

Glycerol stock of :

S39 - pINTE3 (Col E3, AmpR)

S40 - pINTg3p (g3p Term, AmpR)

S41 - pINK2(TolRII, AmpR)

S42 - pTA (pTet-Col A, AmpR)

A28 A29 A28 OmpA signal (ex A11)

A29 FecR mutant

PCR 2 steps : 72°C 30s elongation

Gel verification Ok (OmpAsignal: weak)

Purification Ok

OD : A28=0,82 A29=0,92

Miniprep send to sequencing.

Miniprep DO with miniprep diluted 1/100:

ClyA colonie 3:1,40

ClyA colonie 7:1,60

ClyA colonie 8:0,8

ClyA colonie 10:1,65

The results were transformed into µg/mL

Mix for sequencing:

RFP PCR

RFP PCR using PSB1A3 and:

-O57+O58==>A30=RFP Nter

-O59+058==>A31=RFP

-O59+O60==>A33=RFP Cter

Mix:

H2O:32,5µL

Buffer 5x:10µL

dNTP:1µL

OF(1/10):2,5µL

OR(1/10):2,5µL

Matrice(PSB1A3 1/10):1µL

Phusiong:0,5µL

PCR Check

/100pb/A30/A31/A33/1kb

Digestion

Digestion PSB1A3 in XP