Team:Paris Saclay/Notebook/July/11

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=='''Lab work'''==
=='''Lab work'''==
-
===='''Objective : obtaining obtaining biobricks in PSB3K3'''====
+
===='''Objective : obtaining obtaining biobricks in pSB3K3'''====
===='''1 - Extraction of BBa_J04450 from DH5α'''====
===='''1 - Extraction of BBa_J04450 from DH5α'''====
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===='''Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2'''====
===='''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'''====
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Expected sizes :  
Expected sizes :  
-
*BphR2_Up/VR, VF/BphR2_Down : 1200bp
+
* BphR2_Up/VR, VF/BphR2_Down : 1200bp
{|
{|
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Expected sizes :  
Expected sizes :  
-
*BphA1_Up/VR, VF/BphA1_Down : 500bp
+
* BphA1_Up/VR, VF/BphA1_Down : 500bp
{|
{|
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| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL of DNA Ladder
* Well 1 : 6µL of DNA Ladder
-
* Well 2 to 7 : 5µL of B......  VF/VR primers+1µL of 6X loading dye
+
* Well 2 to 5 : 5µL of BphR1 with VF/VR primers+1µL of 6X loading dye
-
* Well 8 : 6µL of DNA Ladder
+
* Well 6 to 13 : 5µL of BphR2 with VF/VR primers+1µL of 6X loading dye
-
* Well 9 to 16 : 5µL of Bph... with VF/VR primers+1µL of 6X loading dye
+
* Well 14 : 6µL of DNA Ladder
 +
* Well 15 to 22 : 5µL of BphA1 with VF/VR primers+1µL of 6X loading dye
* Well 17 : 6µL of DNA Ladder
* Well 17 : 6µL of DNA Ladder
* Gel : 1.5%
* Gel : 1.5%

Latest revision as of 23:58, 4 October 2013

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Contents

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

[[Psgel11107.jpg]]
  • 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 :

align="center"

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

Abdou, Anaïs

Psgel21107.jpg
  • 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
Psgel31107.jpg
  • 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
Psgel41107.jpg
  • 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
Psgel51107.jpg
  • Well 1 : 6µL of DNA Ladder
  • Well 2 to 5 : 5µL of BphR1 with VF/VR primers+1µL of 6X loading dye
  • Well 6 to 13 : 5µL of BphR2 with VF/VR primers+1µL of 6X loading dye
  • Well 14 : 6µL of DNA Ladder
  • Well 15 to 22 : 5µL of BphA1 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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