Wednesday 30th July

Lab Work

A - The frame coli Odor free

Preparation of electrocompetent cells

by Romain

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

Protocol:

Two dilution of 500µl of bacterial culture MG1655Z1 and E. coli MG1655 in 15ml 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 15ml 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 Romain

Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT340 (code for a flipase for E. coli)

Make 2 electroporations in cold electroporation cuvettes:

A control cuvette(without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
A second cuvette: 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 cuvette and transfer in 2 tubes.

Incubate during 1h at 30°C.

Spread on 8 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.
Transformed and concentrated 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.
Transformed and concentrated E. coli MG1655Z1 with BT340.

Incubate for a night at 30°C.

D - Lemon scent

PCR of BBa_K762100

by Sean

This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into each PCR tube the following:


Component	For a total volume of 50μl
H₂O	35.5μl
Phusion buffer 5X	10μl
dNTPs	1μl
iPS66	1μl
iPS67	1μl
BBa_K762100	1μl
Phusion DNA polymerase	0.25μl

Tube was placed in PCR machine with the following parameters.


Cycle step	Temperature	Time	Cycle
Initial denaturation	98°C	1 min	1
Denaturation	98°C	15 s	25 - 30
Annealing	52°C	20 s	25 - 30
Extension	72°C	45 s	25-30
Final extension	72°C	10 min	1
Final extension	8°C	hold	1

Members there:

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

Back to the calendar

Team:Paris Saclay/Notebook/July/30

From 2014.igem.org

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