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Notebook : August 5

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3

Nadia, XiaoJing

Used quantities :

BBa_K1155003 : 5µL
EcoRI FD : 1µL
PstI FD : 1µL
Buffer FD : 3µL
H2O : 20µL

We incubate the digestion at 37°C during 15 minutes.

2 - Electrophoresis of the digestion of BBa_K1155003

Damir

Well 1 : 6µL of DNA Ladder
Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
Well 3 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
Well 5 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI +6µl of 6X loading dye
Well 7 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
Gel : 1%

Expected sizes :

RBS_AmilCP-Term : 824bp
pSB1C3 : 2070bp

We obtained fragments at the right size. We will sequence it.

Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI

Abdou, Damir, Nadia,

Used quantities :

BBa_I732017 : 41µL
Buffer FD : 5µL
EcoRI : 2µL
SpeI : 2µL

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

2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI

Damir, Nadia

Well 1 : 6µL of DNA Ladder
Well 2 : 40µL of BBa_I732017 digested by EcoRI/Spe I+ 8µL 6X loading dye
Gel : 1.5%

Expected sizes :

RBS-LacZ : 3093 bp
pSB1A2 : 2079 bp

We obtained fragments at the right size. We will make an electro-elution to extract RBS-LacZ.

3 - PCR of pSB1C3

Nadia, XiaoJing

Used quantities :

pSB1C3 : 2µL
Buffer phusion : 10µL
Oligo 64 : 2.5µL
Oligo 65 : 2.5µL
dNTP : 1µL
enzyme Phusion : 0.25µL
H2O : 36.75µL

PCR program :

Hybridation temperature gradient :

A - 65°C/ B - 64.7°C/ C - 64.1°C/ D - 63.1°C/ E - 62°C/ F - 61.2°C/ G - 60.5°C/ H - 60°C/

3 - Electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

Well 1 : 6µL of DNA Ladder
Well 2 to 9 : 2µL of pSB1C3 + 3µL of H2O + 1µL of 6X loading dye
Gel : 1%

Expected sizes :

pSB1C3 : 2070bp

We obtained fragments at the right size. We will purify it.

4 - Gel purification of electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

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

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α

Damir, Nadia, XiaoJing

Transformation of 07/31/13 works. We will do 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 of resuspend colony
Mix PCR :23µL

Total volume: 25µL

PCR Program :

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@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''Objective : obtaining Bba_K1155003'''====
+===='''Objective : obtaining BBa_K1155003'''====
-===='''1 - Digestion of Bba_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and PSB1C3 '''====
+===='''1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3 '''====
 Nadia, XiaoJing
 Used quantities :
-* Bba_K1155003 : 5µL
+* BBa_K1155003 : 5µL
-* EcoRI FD : 1µL
+* EcoRI FD  : 1µL
 * PstI FD : 1µL
 * Buffer FD : 3µL
 * H2O : 20µL
-We let the digestion at 37°C during 15 minutes.
+We incubate the digestion at 37°C during 15 minutes.
-===='''2 - Electrophoresis of the digestion of Bba_K1155003'''====
+===='''2 - Electrophoresis of the digestion of BBa_K1155003'''====
 Damir
 {|
-| style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel10508.jpg| 400px]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-* Well 1 :
+* Well 1 : 6µL of DNA Ladder
-* Well 2 : 6µL of DNA Ladder
+* Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
-* Well 3 : 30µL of Bba_K1155003 from clone 9 digested by EcoRI/PstI+6µl of 6X loading dye
+* Well 3 : 5µL of BBa_K1155003 from clone 9 + 1µl of 6X loading dye
-* Well 4 : 5µL of Bba_K1155003 from clone 9+1µl of 6X loading dye
+* Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI + 6µl of 6X loading dye
-* Well 5 : 30µL of Bba_K1155003 from clone 11 digested by EcoRI/PstI+6µl of 6X loading dye
+* Well 5 : 5µL of BBa_K1155003 from clone 11 + 1µl of 6X loading dye
-* Well 6 : 5µL of Bba_K1155003 from clone 11+1µl of 6X loading dye
+* Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI  +6µl of 6X loading dye
-* Well 7 : 30µL of Bba_K1155003 from clone 12 digested by EcoRI/PstI+6µl of 6X loading dye
+* Well 7 : 5µL of BBa_K1155003 from clone 12 + 1µl of 6X loading dye
-* Well 8 : 5µL of Bba_K1155003 from clone 12+1µl of 6X loading dye
 * Gel : 1%
 |}
 Expected sizes :
-* RBS_Amil CP-Term :
+* RBS_AmilCP-Term : 824bp
-* PSB1C3 :
+* pSB1C3 : 2070bp
-===='''Objective : obtaining Bba_K1155007'''====
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We obtained fragments at the right size. We will sequence it.
+|}
+===='''Objective : obtaining BBa_K1155007'''====
-===='''1 - Digestion of Bba_I732017 by EcoRI/SpeI'''====
+===='''1 - Digestion of BBa_I732017 by EcoRI/SpeI'''====
 Abdou, Damir, Nadia,
 Used quantities :
-* Bba_I732017 : 41µL
+* BBa_I732017 : 41µL
 * Buffer FD : 5µL
 * EcoRI : 2µL
 * SpeI : 2µL
-We let the digestion at 1h30 at 37°C.
+We incubate the digestion  at 37°C for 1h30.
-===='''2 -Electrophoresis to check the digestion of Bba_I732017 by EcoRI/SpeI'''====
+===='''2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI'''====
 Damir, Nadia
 {|
-| style="width:350px;border:1px solid black;" |[[]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel20508.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-* Well 1 : 40µL of Bba_I732017 digested by EcoRI/SpeI+8µL 6X loading dye
+* Well 1 : 6µL of DNA Ladder
+* Well 2 : 40µL of BBa_I732017 digested by EcoRI/Spe I+ 8µL 6X loading dye
 * Gel : 1.5%
 |}
@@ Line 71: / Line 76: @@
 Expected sizes :
 * RBS-LacZ : 3093 bp
-* PSB1A2 : 2079 bp
+* pSB1A2 : 2079 bp
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-We obtain fragments at the right size. We will make an elector-elution to extract RBS-LacZ.
+We obtained fragments at the right size. We will make an electro-elution to extract RBS-LacZ.
 |}
-===='''Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+===='''3 - PCR of pSB1C3'''====
-===='''1 - Colony PCR of Bba_K1155004, Bba_K115005, Bba_K1155006 in DH5α'''====
+Nadia, XiaoJing
+Used quantities :
+* pSB1C3 : 2µL
+* Buffer phusion : 10µL
+* Oligo 64 : 2.5µL
+* Oligo 65 : 2.5µL
+* dNTP : 1µL
+* enzyme Phusion : 0.25µL
+* H2O : 36.75µL
+PCR program :
+Hybridation temperature gradient :
+A - 65°C/
+B - 64.7°C/
+C - 64.1°C/
+D - 63.1°C/
+E - 62°C/
+F - 61.2°C/
+G - 60.5°C/
+H - 60°C/
+[[File:PsPCRC30508.jpg|400px]]
+===='''3 - Electrophoresis of PCR of pSB1C3'''====
+Nadia, XiaoJing
+{|
+| style="width:350px;border:1px solid black;" |[[File:Psgel30508.jpg| 400px]]
+| style="width:350px;border:1px solid black;vertical-align:top;" |
+* Well 1 : 6µL of DNA Ladder
+* Well 2 to 9 : 2µL of pSB1C3 + 3µL of H2O + 1µL of 6X loading dye
+* Gel : 1%
+|}
+Expected sizes :
+* pSB1C3 : 2070bp
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We obtained fragments at the right size. We will purify it.
+|}
+===='''4 - Gel purification of electrophoresis of PCR of pSB1C3'''====
+Nadia, XiaoJing
+Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
+===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
+===='''1 -  PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α'''====
 Damir, Nadia, XiaoJing
@@ Line 89: / Line 148: @@
 |}
-COLONIES PIQUEES DANS 10µL d'eau par Tube !!!!!!!!!!!!!
+We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.
 Used quantities :
-* DNA : 2µL
-* Mix  : (it was divided in 25 tubes for each promotor with 23µL of mix in each tube)
+PCR preparation mix for 25 different colonies including:
 ** Oligo 44 : 3.5µL
 ** Oligo 45 : 3.5µL
@@ Line 100: / Line 159: @@
 ** Dream Taq : 5µL
 ** H2O : 590µL
+PCR reaction:
+* DNA : 2µL of resuspend colony
+* Mix PCR  :23µL
+Total volume: 25µL
 PCR Program :
@@ Line 106: / Line 170: @@
+{| border="1" align="center"
+|[[Team:Paris Saclay/Notebook/August/2|<big>Previous day</big>]]
+|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
+|[[Team:Paris Saclay/Notebook/August/6|<big>Next day</big>]]
+|}
 {{Team:Paris_Saclay/incl_fin}}

Team:Paris Saclay/Notebook/August/5

From 2013.igem.org

Latest revision as of 01:25, 5 October 2013

Contents

Notebook : August 5

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155003

1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and pSB1C3

2 - Electrophoresis of the digestion of BBa_K1155003

Objective : obtaining BBa_K1155007

1 - Digestion of BBa_I732017 by EcoRI/SpeI

2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI

3 - PCR of pSB1C3

3 - Electrophoresis of PCR of pSB1C3

4 - Gel purification of electrophoresis of PCR of pSB1C3

Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - PCR Colonies of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α