Team:Paris Saclay/Notebook/August/8

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Obtaining the PSB3K3 backbone plasmid'''====
+
===='''Obtaining biobricks in pSB3K3'''====
-
=====1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion was right=====
+
====1 - Digestion of BBa_J04450 by EcoRI/PstI====
 +
 
 +
Nadia
 +
 
 +
Used quantities :
 +
* DNA : 5µL
 +
* Buffer FD : 2µL
 +
* EcoRI FD : 1µL
 +
* PstI FD : 1µL
 +
* H2O : 11µL
 +
 
 +
We incubate our digestion  at 37°C for 1h30.
 +
 
 +
====2 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion====
Damir, Nadia
Damir, Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" | IMAGE
+
| style="width:350px;border:1px solid black;" |[[File:Psgel10808.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
Line 22: Line 35:
Expected sizes :
Expected sizes :
-
*PSB3K3 : 2750kb
+
*pSB3K3 : 2750kb
*GFP : 1069kb
*GFP : 1069kb
-
We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.
 +
|}
-
=====2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1=====
+
====3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI====
-
 
+
-
''2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1''
+
Anaïs
Anaïs
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_eelution.jpg|350px]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel20808.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
-
*Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
+
*Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst I
*Gel : 0.8%
*Gel : 0.8%
|}
|}
-
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
+
pSB3K3 : 2750bp
-
''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.
 +
|}
 +
 
 +
==== 4- Electroelution of pSB3K3 digested by EcoRI/PstI====
Nadia
Nadia
-
Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
+
Protocol : [[Team:Paris_Saclay/electro|Electroelution]]
-
We let the plasmid precipitate during the night.
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost our plasmid. We will do the digestion again.
 +
|}
-
===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
+
===='''Objective : obtaining BBa_K1155007'''====
 +
 
 +
====1 - Colony PCR of BBa_K115007 in DH5α====
-
=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
 
Anaïs
Anaïs
-
*Colony counting :
+
{|
-
**Low concentration petri dish : 47 colonies
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
**High concentration petri dish : 145 colonies
+
Transformation of 08/07/13 works. we will make a PCR Colony.
 +
|}
-
*Picking of 25 colonies
+
We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.
-
*Preparation of 700µL of Master mix
+
Used quantities :
-
**H<sub>2</sub>O : 590µL
+
-
**dNTP : 28µL
+
-
**VF2 primer : 3.5µL
+
-
**VR primer : 3.5µL
+
-
**DreamTaq buffer 10x : 70µL
+
-
**DreamTaq enzyme : 5µL
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
+
PCR preparation mix for 25 different colonies including:
 +
** Oligo 44 : 3.5µL
 +
** Oligo 45 : 3.5µL
 +
** Buffer Dream Taq : 70µL
 +
** dNTP : 28µL
 +
** Dream Taq : 5µL
 +
** H2O : 590µL
 +
 
 +
PCR reaction: 
 +
* DNA : 2µL
 +
* Mix  :23µL
 +
Total volume: 25µL
PCR Program :
PCR Program :
Line 76: Line 105:
[[File:PsPcr808.jpg|400px]]
[[File:PsPcr808.jpg|400px]]
-
=====2 - Gel electrophoresis of the colony PCR products=====
+
====2 - Electrophoresis to check the colony PCR products : BBa_K1155007====
 +
 
Anaïs, Damir
Anaïs, Damir
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel30808.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*6µL DNA Ladder
+
* Well 1 : 6µL DNA Ladder
-
*10µL sample per well
+
* Well 2 to 24 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
 +
* Well 25 : 6µL DNA Ladder
 +
* Well 26 : 6µL DNA Ladder
 +
* Well 27 : 6µL DNA Ladder
 +
* Well 28 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
*Gel : 0.8%
*Gel : 0.8%
|}
|}
-
Expected size : 3583bp
+
Expected size :  
 +
* BBa_K1155007 : 3583 bp
-
Colonies 10, 14, 15 exhibit plasmids with the right length.
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007.
 +
|}
 +
 
 +
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
 +
 
 +
===='''1 - Tranduction of Km in MG1655Z1 ====
 +
 
 +
Anaïs, Nadia
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
We didn't obtain colonies from the transduction of 08/07/13. We will do it again.
 +
|}
 +
 
 +
Protocol : [[Team:Paris_Saclay/transduction|Transduction]]
 +
 
 +
Our Mutant strain is BW1328 (Δfnr::Km) and the wild type strain is MG1655Z1.
 +
 
 +
We did the first step of the protocol : bacteriophage stock on BW1328 (Δfnr::Km).
 +
 
 +
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
 +
 
 +
===='''Objective : obtaining FNR and BphR2 proteins'''====
 +
 
 +
====1 - Extraction of plasmid  pSB1C3 containing BphR2 ,FNR and RBS-FNR in competent cell DH5α====
-
====3 - PCR product (made the 08/01/2013) purification====
 
Damir
Damir
-
available quantity:
+
{|
-
* FNR Part1 : 10 µl
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
* FNR Part2 : 19 µl
+
Transformation of 08/02/13 works. We will extract plasmids.
-
* RBS FNR Part1 :16.1µl
+
|}
-
* RBS BphR2 Part1 : 28µl
+
-
* BphR2 Part1 : 16.4 µl
+
-
* BphR2 Part2 : 18.9 µl
+
-
Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
-
<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost our plasmids. We will do the Gibson assembly again.
 +
|}
-
+
====2 - Gel purification of RBS-BphR2 Part I====
 +
Nadia, XiaoJing
 +
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost fragment. We will do the PCR again.
 +
|}
 +
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/7|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/9|<big>Next day</big>]]
 +
|}
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Latest revision as of 01:26, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining biobricks in pSB3K3

1 - Digestion of BBa_J04450 by EcoRI/PstI

Nadia

Used quantities :

  • DNA : 5µL
  • Buffer FD : 2µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL
  • H2O : 11µL

We incubate our digestion at 37°C for 1h30.

2 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion

Damir, Nadia

Psgel10808.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8%

Expected sizes :

  • pSB3K3 : 2750kb
  • GFP : 1069kb

We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.

3 - Electrophoresis of BBa_J004450 digested by EcoRI/PstI

Anaïs

Psgel20808.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst I
  • Gel : 0.8%

pSB3K3 : 2750bp

We obtained fragments at the right size. We will purify the highest band which contains the pSB3K3 plasmid.

4- Electroelution of pSB3K3 digested by EcoRI/PstI

Nadia

Protocol : Electroelution

We lost our plasmid. We will do the digestion again.

Objective : obtaining BBa_K1155007

1 - Colony PCR of BBa_K115007 in DH5α

Anaïs

Transformation of 08/07/13 works. we will make a PCR Colony.

We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.

Used quantities :

PCR preparation mix for 25 different colonies including:

    • Oligo 44 : 3.5µL
    • Oligo 45 : 3.5µL
    • Buffer Dream Taq : 70µL
    • dNTP : 28µL
    • Dream Taq : 5µL
    • H2O : 590µL

PCR reaction:

  • DNA : 2µL
  • Mix  :23µL

Total volume: 25µL

PCR Program :

PsPcr808.jpg

2 - Electrophoresis to check the colony PCR products : BBa_K1155007

Anaïs, Damir

Psgel30808.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 24 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
  • Well 25 : 6µL DNA Ladder
  • Well 26 : 6µL DNA Ladder
  • Well 27 : 6µL DNA Ladder
  • Well 28 : 10µL of BBa_K1155007 + 2µL of 6X loading dye
  • Gel : 0.8%

Expected size :

  • BBa_K1155007 : 3583 bp

We obtained fragments at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007.

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Tranduction of Km in MG1655Z1

Anaïs, Nadia

We didn't obtain colonies from the transduction of 08/07/13. We will do it again.

Protocol : Transduction

Our Mutant strain is BW1328 (Δfnr::Km) and the wild type strain is MG1655Z1.

We did the first step of the protocol : bacteriophage stock on BW1328 (Δfnr::Km).

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - Extraction of plasmid pSB1C3 containing BphR2 ,FNR and RBS-FNR in competent cell DH5α

Damir

Transformation of 08/02/13 works. We will extract plasmids.

Protocol : Gel purification

We lost our plasmids. We will do the Gibson assembly again.

2 - Gel purification of RBS-BphR2 Part I

Nadia, XiaoJing

Protocol : Gel purification

We lost fragment. We will do the PCR again.


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