From 2013.igem.org

(Difference between revisions)

Latest revision as of 00:29, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Notebook : July 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP

Abdou

Protocol : Ethanol precipitation

We used 90µL of DNA. At the end, we mix our DNA with 20µL of water.

2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP

Abdou

Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye
Well 2 : 6µL of DNA Ladder
Gel : 1%

Expected sizes :

RBS-AmilCP : 3500bp

We obtain fragments at the right size. We will ligate RBS-AmilCP with Term.

3 - Ligation of RBS-AmilCP and Term-pSB1C3

Xavier

Used quantities :

RBS-AmilCP : 2µL
Term in pSB1C3 : 2.5µL
Buffer : 1µL
Ligase : 1µL
H2O : 3.5µL

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou

Well 1 : 5µL of NirB+1µL of 6X loading dye
Well 2 : 5µL of NirB+1µL of 6X loading dye
Well 3 : 5µL of NarG+1µL of 6X loading dye
Well 4 : 5µL of NarG+1µL of 6X loading dye
Well 5 : 6µL DNA Ladder
Well 6 : 5µL of NarK+1µL of 6X loading dye
Well 7 : 5µL of NarK+1µL of 6X loading dye
Well 8 : 5µL of NarK+1µL of 6X loading dye
Gel : 1%

Expected sizes :

NarK, NarG, NirB : 200bp

We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos.

2 - PCR of NarK, NarG, NirB

Abdou, Xavier

Used quantities :

NarK :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 47 : 2µL
- Oligo 48 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL

NarG :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 49 : 2µL
- Oligo 50 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL

NirB :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo 45 : 2µL
- Oligo 46 : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL

PCR Program :

3 - Electrophoresis of PCR products : NarK, NarG, NirB

Abdou, Xavier

Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
Well 2 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
Well 3 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
Well 4 : 5µL of NarK+3µL H2O+1µL of 6X loading dye
Well 5 : 6µL DNA Ladder
Well 6 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
Well 7 : 5µL of NarG+3µL H2O+1µL of 6X loading dye
Gel : 1.2%

Expected sizes :

NarK, NarG, NirB : 200bp

We obtain fragments at the right size. We can purify it.

4 - Gel purification of PCR products : NarK, NarG, NirB

Xavier

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

At the end we mix our DNA in 20µL of H2O.

5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI

Abdou

Quantities used :

NarK, NarG, NirB : 10µL
Buffer FD : 3µL
EcoRI FD : 1.5µL
PstI FD : 1.5µL
H2O : 14µL

We let the digestion at 37°C during 1h30 and then at -20°C.

B - PCB sensor system

Objective : obtaining BBa_K1155002

1 - Tranformation of BBa_K1155002 in DH5α

Abdou, Xavier

Protocol : Bacterial tranformation

Previous day

Back to calendar

Next day

@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''Objective : obtaining Bba_K1155003'''====
+===='''Objective : obtaining BBa_K1155003'''====
-===='''1 - Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''====
+===='''1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP '''====
 Abdou
@@ Line 16: / Line 16: @@
 We used 90µL of DNA.
-On a reprit dans 20µL d'eau !!!!!!!!!!!!!!!!!!!!!!!!
+At the end, we mix our DNA with 20µL of water.
-===='''2 - Ligation of RBS-AmilCP and Term-PSB1C3'''====
+===='''2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP'''====
+Abdou
+{|
+| style="width:350px;border:1px solid black;" |[[File:Psgel2607.jpg]]
+| style="width:350px;border:1px solid black;vertical-align:top;" |
+* Well 1 : 5µL of RBS-AmilCP+1µL of 6X loading dye
+* Well 2 : 6µL of DNA Ladder
+* Gel : 1%
+|}
+Expected sizes :
+* RBS-AmilCP : 3500bp
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We obtain fragments at the right size. We will ligate RBS-AmilCP with Term.
+|}
+===='''3 - Ligation of RBS-AmilCP and Term-pSB1C3'''====
 Xavier
@@ Line 25: / Line 45: @@
 * RBS-AmilCP : 2µL
-* Term in PSB1C3 : 2.5µL
+* Term in pSB1C3 : 2.5µL
 * Buffer : 1µL
 * Ligase : 1µL
 * H2O :   3.5µL
-===='''Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
 ===='''1 - Electrophoresis of PCR products : NarK, NarG, NirB'''====
@@ Line 37: / Line 57: @@
 {|
-| style="width:350px;border:1px solid black;" |[[]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel12607.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
 * Well 1 : 5µL of NirB+1µL of 6X loading dye
-* Well 2 : 5µL of NirB+1µL of 6X loading dye LA MEME CHOSE ???????
+* Well 2 : 5µL of NirB+1µL of 6X loading dye
 * Well 3 : 5µL of NarG+1µL of 6X loading dye
 * Well 4 : 5µL of NarG+1µL of 6X loading dye
 * Well 5 : 6µL DNA Ladder
-* Well 6 : 5µL of NarK+1µL of 6X loading dye NUMERO 4 ???????????????
+* Well 6 : 5µL of NarK+1µL of 6X loading dye
 * Well 7 : 5µL of NarK+1µL of 6X loading dye
 * Well 8 : 5µL of NarK+1µL of 6X loading dye
@@ Line 51: / Line 71: @@
 Expected sizes :
-* NarK, NarG, NirB : ...
+* NarK, NarG, NirB : 200bp
 {|
@@ Line 67: / Line 87: @@
 ** Buffer phusion : 10µL
 ** dNTP : 1µL
-** Oligo ... : 2µL
+** Oligo 47 : 2µL
-** Oligo ... : 2µL
+** Oligo 48 : 2µL
 ** Concentrated DNA : 2µL
 ** Phusion : 0.25µL
@@ Line 75: / Line 95: @@
 ** Buffer phusion : 10µL
 ** dNTP : 1µL
-** Oligo ... : 2µL
+** Oligo 49 : 2µL
-** Oligo ... : 2µL
+** Oligo 50 : 2µL
 ** Concentrated DNA : 2µL
 ** Phusion : 0.25µL
@@ Line 83: / Line 103: @@
 ** Buffer phusion : 10µL
 ** dNTP : 1µL
-** Oligo ... : 2µL
+** Oligo 45 : 2µL
-** Oligo ... : 2µL
+** Oligo 46 : 2µL
 ** Concentrated DNA : 2µL
 ** Phusion : 0.25µL
@@ Line 97: / Line 117: @@
 {|
-| style="width:350px;border:1px solid black;" |[[]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel22607.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
 * Well 1 : 2µL of NirB+3µL H2O+1µL of 6X loading dye
@@ Line 110: / Line 130: @@
 Expected sizes :
-* NarK, NarG, NirB : ...
+* NarK, NarG, NirB : 200bp
 {|
@@ Line 123: / Line 143: @@
 Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
-REPRIS dans 20µL !!!!!!!!
+At the end we mix our DNA in 20µL of H2O.
 ===='''5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI'''====
@@ Line 142: / Line 162: @@
 ==='''B - PCB sensor system'''===
-===='''Objective : obtaining Bba_K1155002'''====
+===='''Objective : obtaining BBa_K1155002'''====
-===='''1 - Tranformation of Bba_K1155002 in DH5α '''====
+===='''1 - Tranformation of BBa_K1155002 in DH5α '''====
 Abdou, Xavier
@@ Line 155: / Line 175: @@
 |[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
-|[[Team:Paris Saclay/Notebook/July/27|<big>Next day</big>]]
+|[[Team:Paris Saclay/Notebook/July/30|<big>Next day</big>]]
 |}
 {{Team:Paris_Saclay/incl_fin}}

Team:Paris Saclay/Notebook/July/26

From 2013.igem.org

Latest revision as of 00:29, 5 October 2013

Contents

Notebook : July 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP

2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP

3 - Ligation of RBS-AmilCP and Term-pSB1C3

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Electrophoresis of PCR products : NarK, NarG, NirB

2 - PCR of NarK, NarG, NirB

3 - Electrophoresis of PCR products : NarK, NarG, NirB

4 - Gel purification of PCR products : NarK, NarG, NirB

5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI

B - PCB sensor system

Objective : obtaining BBa_K1155002

1 - Tranformation of BBa_K1155002 in DH5α