Revision as of 16:32, 28 July 2014

Monday 28th July

Lab work

A - The frame

Preparation of electrocompetent cells

by Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655.

Protocol:

Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 30ml of LB at 30°C for each strain.

When the culture OD₆₅₀ = 0,6:

put in ice during 10min.
centriguge at 4°C, 5min, 4000rpm.
Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% COLD.
centriguge at 4°C, 5min, 4000rpm.
Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
centriguge at 4°C, 5min, 4000rpm.
Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.

Transformation of electrocompetent cells

by Arnaud & Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT640 (code for a flipase for E. coli)

Make 2 électroporations in cold electroporation tanks:

A control tank (without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
A second tank: 50µl of E. coli MG1655Z1 or E. coli MG1655 culture + 1µl of BT640 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each tank and transfer in 2 tubes.

Spread on 4 dishes LB + Cm:

20µl of control E. coli MG1655Z1 (without plasmid)
50µl of transformed E. coli MG1655Z1 with BT340.
100µl of transformed E. coli MG1655Z1 with BT340.
Concentrate of transformed E. coli MG1655Z1 with BT340.

20µl of control E. coli MG1655 (without plasmid)
50µl of transformed E. coli MG1655 with BT340.
100µl of transformed E. coli MG1655 with BT340.
Concentrate of transformed E. coli MG1655Z1 with BT340.

Incubate for a night at 30°C.

C - Lemon scent

Liquid Culture

by Fabio

Due to an widespread infection of the cells made the 25th July, we collected the original strain DY330 to verify it's legitimacy.

2ml LB + 20μl of DY330. We incubate cultures at 30°C.

E - Salicylate Inducible Suppressing System

Plasmid DNA Purification

by Fabio

BBa_J61051
BBa_K228001

from Bacterial Culture made the 25th July

Protocol

Members present:

Instructors and advisors: Solenne and Sylvie.
Students: Arnaud, Fabio, Romain, Sean and Terry.

Back to the calendar

@@ Line 2: / Line 2: @@
 ==Lab work==
+===A - The frame===
+====Preparation of electrocompetent cells====
+''by Arnaud & Romain''
+Strain used: E. coli MG1655Z1 and E. coli MG1655.
+Protocol:
+Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 30ml of LB at 30°C for each strain.
+When the culture OD<sub>650</sub> = 0,6:
+*put in ice during 10min.
+*centriguge at 4°C, 5min, 4000rpm.
+*Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% '''COLD'''.
+*centriguge at 4°C, 5min, 4000rpm.
+*Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''.
+*centriguge at 4°C, 5min, 4000rpm.
+*Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% '''COLD'''.
+====Transformation of electrocompetent cells====
+''by Arnaud & Romain''
+Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT640 (code for a flipase for E. coli)
+Make 2 électroporations in cold electroporation tanks:
+*A control tank (without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
+*A second tank: 50µl of E. coli MG1655Z1 or E. coli MG1655 culture + 1µl of BT640 plasmid.
+Electroporation : 2500V, 132W, 40µF.
+After that, add 1ml of cold LB in each tank and transfer in 2 tubes.
+Spread on 4 dishes LB + Cm:
+*20µl of control E. coli MG1655Z1 (without plasmid)
+*50µl of transformed E. coli MG1655Z1 with BT340.
+*100µl of transformed E. coli MG1655Z1 with BT340.
+*Concentrate of transformed E. coli MG1655Z1 with BT340.
+*20µl of control E. coli MG1655 (without plasmid)
+*50µl of transformed E. coli MG1655 with BT340.
+*100µl of transformed E. coli MG1655 with BT340.
+*Concentrate of transformed E. coli MG1655Z1 with BT340.
+Incubate for a night at 30°C.
 ===C - Lemon scent===

Team:Paris Saclay/Notebook/July/28

From 2014.igem.org