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Notebook : July 11

Lab work

Objective : obtaining obtaining biobricks in PSB3K3

1 - Extraction of BBa_J04450 from DH5α

Sheng

Mini and maxi preparation of 07/10/13 works. We will extract DNA.

Protocol : Low copy plamid extraction

2 - Digestion of BBa_J04450 to chek the size for the plasmid

Sheng

Used quantities :

XhoI :
- DNA : 3µL
- Buffer Green : 3µL
- XhoI : 1µL
- H2O : 21µL

SacII :
- DNA : 3µL
- Buffer Blue : 3µL
- SacII : 1µL
- H2O : 21µL

EcoRI/PstI :
- DNA : 5µL
- Buffer Orange : 3µL
- EcoRI : 1µL
- PstI : 1µL
- H2O : 20µL

XhoI/SacII :
- DNA : 5µL
- Buffer Green : 3µL
- XhoI : 1µL
- SacII : 1µL
- H2O : 20µL

3 - Electrophoresis of the digestion of BBa_J04450

Zhou

[[

]]

Well 1 : 20µL of pSB3K3 digested by XhoI/SacII+4µL of 6X loading dye
Well 2 : 20µL of pSB3K3 digested by SacII+4µL of 6X loading dye
Well 3 : 20µL of pSB3K3 digested by EcoRI/PstI+4µL of 6X loading dye
Well 4 : 20µL of pSB3K3 digested by XhoI+4µL of 6X loading dye
Well 5 : 2µL of pSB3K3+1µL of 6X loading dye
Well 6 : 6µL of DNA Ladder
Well 7 : 20µL of pSB3K3 digested by XhoI/SacII+4µL of 6X loading dye
Well 8 : 20µL of pSB3K3 digested by SacII+4µL of 6X loading dye
Well 9 : 20µL of pSB3K3 digested by EcoRI/PstI+4µL of 6X loading dye
Well 10 : 20µL of pSB3K3 digested by XhoI+4µL of 6X loading dye
Well 11 : 2µL of pSB3K3+1µL of 6X loading dye
Gel : 1.5%

Expected size

pSB3K3 : 3819 bp
EcoRI/PstI : 1069 bp + 2750 bp
XhoI : 2976 bp + 843 bp
SacII : 3819 bp
XhoI/SacII : 843 bp + 616 bp + 2367 bp

We obtain fragments at the right size for pSB3K3, EcoRI/PstI digestion and SacII digestion. Digestoins with XhoI didn't seem to work. The extraction of BBa_J04450 in DH5α was good.

B - PBC sensor system

Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2

1 - Colony PCR of BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3 in DH5α to check good insertions of BphR1, BphA1, BphR2 in pSB1C3

Abdou, Anaïs, Zhou

Transformation of BBa_K1155001, BBa_K1155002 and BphR2 protein in DH5α of 07/10/13 work. We will do a Colony PCR.

We mix our colonies in 10µL of H2O.

Used quantities :

DNA : 2µL

Mix A : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphR1_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix B : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphR1_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix C : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphR2_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix D : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphR2_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix E : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- BphA1_Up/VR : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix F : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 50µL
- MgCl2 : 20µL
- dNTP : 10µL
- VF/BphA1_Down : 1.25µL for each oligo
- Enzyme : 2.5µL
- H2O : 145µL

Mix G : (it was divided in 8tubes for 8 colonies different)
- Buffer Go Taq : 125µL
- MgCl2 : 50µL
- dNTP : 25µL
- VF/VR: 3µL for each oligo
- Enzyme : 6.25µL
- H2O : 362.75µL

PCR program :

2 - Electrophoresis of Colony PCR products : BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3

Abdou, Anaïs

Well 1 : 6µL of DNA Ladder
Well 2 to 9 : 5µL of BphR1 BphR1_Up/VR primers+1µL of 6X loading dye
Well 10 : 6µL of DNA Ladder
Well 11 to 18 : 5µL of BphR1 with VF/BphR1_Down primers+1µL of 6X loading dye
Well 19 : 6µL of DNA Ladder
Gel : 1.5%

Expected sizes :

BphR1_Up/VR, VF/BphR1_Down : 500bp

Well 1 : 6µL of DNA Ladder
Well 2 to 9 : 5µL of BphR2 BphR2_Up/VR primers+1µL of 6X loading dye
Well 10 : 6µL of DNA Ladder
Well 11 to 18 : 5µL of BphR2 with VF/BphR2_Down primers+1µL of 6X loading dye
Well 19 : 6µL of DNA Ladder
Gel : 1.5%

Expected sizes :

BphR2_Up/VR, VF/BphR2_Down : 1200bp

Well 1 : 6µL of DNA Ladder
Well 2 to 9 : 5µL of BphA1 BphA1_Up/VR primers+1µL of 6X loading dye
Well 10 and 15 : 6µL of DNA Ladder
Well 16 to 23 : 5µL of BphA1 with VF/BphA1_Down primers+1µL of 6X loading dye
Gel : 1.5%

Expected sizes :

BphA1_Up/VR, VF/BphA1_Down : 500bp

Well 1 : 6µL of DNA Ladder
Well 2 to 7 : 5µL of B...... VF/VR primers+1µL of 6X loading dye
Well 8 : 6µL of DNA Ladder
Well 9 to 16 : 5µL of Bph... with VF/VR primers+1µL of 6X loading dye
Well 17 : 6µL of DNA Ladder
Gel : 1.5%

Expected sizes :

BphR2 : 1200bp
BphR1, BphA1 : 500bp

We didn't obtain fragments at the right size but we will do streaking of clone 5, 6 for BphR1, clone 5, 6, 7, 8 for BphA1, clones 3, 4 for BphR2.

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@@ Line 56: / Line 56: @@
 | style="width:350px;border:1px solid black;" |[[[[File:Psgel11107.jpg|500px]]]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-* Well 1 : 20µL of PSB3K3 digested by XhoI/SacII+4µL of  6X loading dye
+* Well 1 : 20µL of pSB3K3 digested by XhoI/SacII+4µL of  6X loading dye
-* Well 2 : 20µL of PSB3K3 digested by SacII+4µL of  6X loading dye
+* Well 2 : 20µL of pSB3K3 digested by SacII+4µL of  6X loading dye
-* Well 3 : 20µL of PSB3K3 digested by EcoRI/PstI+4µL of  6X loading dye
+* Well 3 : 20µL of pSB3K3 digested by EcoRI/PstI+4µL of  6X loading dye
-* Well 4 : 20µL of PSB3K3 digested by XhoI+4µL of  6X loading dye
+* Well 4 : 20µL of pSB3K3 digested by XhoI+4µL of  6X loading dye
-* Well 5 : 2µL of PSB3K3+1µL of 6X loading dye
+* Well 5 : 2µL of pSB3K3+1µL of 6X loading dye
 * Well 6 : 6µL of DNA Ladder
-* Well 7 : 20µL of PSB3K3 digested by XhoI/SacII+4µL of  6X loading dye
+* Well 7 : 20µL of pSB3K3 digested by XhoI/SacII+4µL of  6X loading dye
-* Well 8 : 20µL of PSB3K3 digested by SacII+4µL of  6X loading dye
+* Well 8 : 20µL of pSB3K3 digested by SacII+4µL of  6X loading dye
-* Well 9 : 20µL of PSB3K3 digested by EcoRI/PstI+4µL of  6X loading dye
+* Well 9 : 20µL of pSB3K3 digested by EcoRI/PstI+4µL of  6X loading dye
-* Well 10 : 20µL of PSB3K3 digested by XhoI+4µL of  6X loading dye
+* Well 10 : 20µL of pSB3K3 digested by XhoI+4µL of  6X loading dye
-* Well 11 : 2µL of PSB3K3+1µL of 6X loading dye
+* Well 11 : 2µL of pSB3K3+1µL of 6X loading dye
 * Gel : 1.5%
 |}
 Expected size
-* PSB3K3 : 3819 bp
+* pSB3K3 : 3819 bp
 * EcoRI/PstI : 1069 bp + 2750 bp
 * XhoI : 2976 bp + 843 bp
@@ Line 79: / Line 79: @@
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-We obtain fragments at the right size for PSB3K3, EcoRI/PstI digestion and SacII digestion. Digestoins with XhoI didn't seem to work. The extraction of BBa_J04450 in DH5α was good.
+We obtain fragments at the right size for pSB3K3, EcoRI/PstI digestion and SacII digestion. Digestoins with XhoI didn't seem to work. The extraction of BBa_J04450 in DH5α was good.
 |}
@@ Line 87: / Line 87: @@
 ===='''Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2'''====
+===='''1 - Colony PCR of BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3 in DH5α to check good insertions of BphR1, BphA1, BphR2 in pSB1C3'''====
-===='''1 - Colony PCR of BBa_K1155001, BBa_K1155002 and BphR2 in PSB1C3 in DH5α to check good insertions of BphR1, BphA1, BphR2 in PSB1C3'''====
 Abdou, Anaïs, Zhou
@@ Line 162: / Line 161: @@
 [[File:PSPCR1107p.jpg|align="center"|400px]]
-===='''2 - Electrophoresis of Colony PCR products : BBa_K1155001, BBa_K1155002 and BphR2 in PSB1C3'''====
+===='''2 - Electrophoresis of Colony PCR products : BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3'''====
 Abdou, Anaïs

Team:Paris Saclay/Notebook/July/11

From 2013.igem.org

Revision as of 16:39, 4 October 2013

Contents

Notebook : July 11

Lab work

Objective : obtaining obtaining biobricks in PSB3K3

1 - Extraction of BBa_J04450 from DH5α

2 - Digestion of BBa_J04450 to chek the size for the plasmid

3 - Electrophoresis of the digestion of BBa_J04450

B - PBC sensor system

Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2

1 - Colony PCR of BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3 in DH5α to check good insertions of BphR1, BphA1, BphR2 in pSB1C3

2 - Electrophoresis of Colony PCR products : BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3