Team:Paris Saclay/Notebook/July/30

From 2014.igem.org

(Difference between revisions)
(Created page with "=Wednesday 30th July= ==Lab Work==")
m (→Wednesday 30th July)
 
(16 intermediate revisions not shown)
Line 1: Line 1:
 +
{{Team:Paris_Saclay/notebook_header}}
=Wednesday 30th July=
=Wednesday 30th July=
-
 
==Lab Work==
==Lab Work==
 +
===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 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 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.
 +
 +
===Salicylate Inducible Suppressing System===
 +
====DNA purification gel agarose====
 +
''by Fabio''
 +
 +
Once the segregate process made [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/29#Electrophoresis yesterday] by electrophoresis, the DNA correspondent to the core of '''BBa_J61051''' was purified from the gel agarose.
 +
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Segregate_process Segregate Process Protocol]
 +
 +
====Ligation====
 +
''by Fabio''
 +
 +
The final step to have a BioBrick Assembly is the Ligation reaction.
 +
 +
<font color="#F00">TODO: illustration of the process</font>
 +
 +
* BioBrick '''BBa_J61051''' (Salicylate promoter + NahR) as '''Part A'''
 +
* BioBrick '''BBa_K228001''' (RNA suppressor) as '''Part B'''
 +
 +
[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Ligation BioBrick Assembly - Ligation Protocol]
 +
 +
===Lemon scent===
 +
====PCR of BBa_K762100====
 +
''by Sean''
 +
 +
This was a second attempt at [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/29 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:
 +
 +
{| class="wikitable centre" width="50%"
 +
|+
 +
|-
 +
! scope=col | Component
 +
! scope=col | For a total volume of 50μl
 +
|-
 +
|H<sub>2</sub>O
 +
|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.
 +
 +
{| class="wikitable centre" width="80%"
 +
|+
 +
|-
 +
! scope=col | Cycle step
 +
! scope=col | Temperature
 +
! scope=col | Time
 +
! scope=col | Cycle
 +
|-
 +
| width="25%" |
 +
Initial denaturation
 +
| width="25%" |
 +
98°C
 +
| width="25%" |
 +
1 min
 +
| width="25%" |
 +
1
 +
|-
 +
| Denaturation
 +
| 98°C
 +
| 15 s
 +
| 25 - 30
 +
|-
 +
| Annealing
 +
| 52°C
 +
| 25 s
 +
| 25 - 30
 +
|-
 +
| Extension
 +
| 72°C
 +
| 45 s
 +
| 25-30
 +
|-
 +
| Final extension
 +
| 72°C
 +
| 10 min
 +
| 1
 +
|-
 +
| Final extension
 +
| 8°C
 +
| hold
 +
| 1
 +
|}
 +
 +
 +
==Photo of the Day==
 +
[[File:Paris Saclay 30_july.jpg|600px|center]]
 +
 +
'''Members there''':
 +
* Instructors and advisors: Alice, Solenne and Sylvie.
 +
* Students: Arnaud, Fabio, Romain, Sean and Terry.
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:44, 14 October 2014

Contents

Wednesday 30th July

Lab Work

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.

Salicylate Inducible Suppressing System

DNA purification gel agarose

by Fabio

Once the segregate process made yesterday by electrophoresis, the DNA correspondent to the core of BBa_J61051 was purified from the gel agarose.

Segregate Process Protocol

Ligation

by Fabio

The final step to have a BioBrick Assembly is the Ligation reaction.

TODO: illustration of the process

  • BioBrick BBa_J61051 (Salicylate promoter + NahR) as Part A
  • BioBrick BBa_K228001 (RNA suppressor) as Part B

BioBrick Assembly - Ligation Protocol

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 25 s 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1


Photo of the Day

Paris Saclay 30 july.jpg

Members there:

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

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/July/30"