Team:Paris Saclay/Notebook/July/29

From 2014.igem.org

(Difference between revisions)
(→PCR of BBa_K762100)
(→Tuesday 29th July)
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==Lab Work==
==Lab Work==
-
===A - The frame===
+
===A - The frame coli Odor free===
====PCR====
====PCR====
''by Romain''
''by Romain''
-
StrainS used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.
+
Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.
Protocol:
Protocol:
Line 30: Line 30:
#Tube 7: MG1655Z1 10I positive control with plasmid
#Tube 7: MG1655Z1 10I positive control with plasmid
-
====Electrophoresis====
+
Results of the PCR: The electrophoresis revealed nothing
-
''by Romain''
+
 
 +
New PCR with different enyme.
 +
 
 +
Strains used: MG1655 and MG1655Z1, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.
 +
 
 +
'''Protocol'''
 +
 
 +
Add into a 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
 +
|37.25μl
 +
|-
 +
| DreamTaq buffer 10X
 +
| 5μl
 +
|-
 +
| dNTPs
 +
| 1μl
 +
|-
 +
| FTR-Apra-F
 +
| 1μl
 +
|-
 +
| FTR-Apra-R
 +
| 1μl
 +
|-
 +
| DMSO
 +
| 2.5μl
 +
|-
 +
| Bacterial culture
 +
| 2μl
 +
|-
 +
| DreamTaq enzyme
 +
| 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%" |
 +
95°C
 +
| width="25%" |
 +
5 min
 +
| width="25%" |
 +
1
 +
|-
 +
| Denaturation
 +
| 95°C
 +
| 30 s
 +
| 30
 +
|-
 +
| Annealing
 +
| 60°C
 +
| 30 s
 +
| 30
 +
|-
 +
| Extension
 +
| 72°C
 +
| 2 min
 +
| 30
 +
|-
 +
| Final extension
 +
| 72°C
 +
| 5 min
 +
| 1
 +
|-
 +
| Final extension
 +
| 12°C
 +
| hold
 +
| 1
 +
|}
 +
 
-
===C - Lemon scent===
+
===D - Lemon scent===
====PCR of BBa_K762100====
====PCR of BBa_K762100====

Revision as of 07:56, 31 July 2014

Contents

Tuesday 29th July

Lab Work

A - The frame coli Odor free

PCR

by Romain

Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.

Protocol:

  • 218µl of H20 MilliQ
  • 80µl GoTaq buffer 5X
  • 16µl oligonucleotide FTR-Apra-F (10mM)
  • 16µl oligonucleotide FTR-Apra-R (10mM)
  • 12µl DMSO 10mM
  • 32µl MgCl2 (25mM)
  • 8µl dNTP (10mM)
  • 2µl of GoTaq enzyme
  • 2µl of different bacterial cultures in each tubes:
  1. Tube 1: MG1655Z1 10I
  2. Tube 2: MG1655 100II
  3. Tube 3: MG1655 50I
  4. Tube 4: MG1655 100I
  5. Tube 5: MG1655Z1 10II
  6. Tube 6: MG1655 50II
  7. Tube 7: MG1655Z1 10I positive control with plasmid

Results of the PCR: The electrophoresis revealed nothing

New PCR with different enyme.

Strains used: MG1655 and MG1655Z1, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.

Protocol

Add into a PCR tube the following:

Component For a total volume of 50μl
H2O 37.25μl
DreamTaq buffer 10X 5μl
dNTPs 1μl
FTR-Apra-F 1μl
FTR-Apra-R 1μl
DMSO 2.5μl
Bacterial culture 2μl
DreamTaq enzyme 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

95°C

5 min

1

Denaturation 95°C 30 s 30
Annealing 60°C 30 s 30
Extension 72°C 2 min 30
Final extension 72°C 5 min 1
Final extension 12°C hold 1


D - Lemon scent

PCR of BBa_K762100

by Sean

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into a 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 55°C variable 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1

E - Salicylate Inducible Suppressing System

Digestion

by Fabio

BioBrick BBa_J61051 (Salicylate promoter + NahR)

Electrophoresis

by Fabio

Results:

First process to verify the digestion

Second one to separate both parts (only with J61051).

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/29"