Team:Paris Saclay/Notebook/July/11

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
 +
='''Notebook : July 11'''=
='''Notebook : July 11'''=
 +
=='''Lab work'''==
-
==''Summary:''==
+
===='''Objective : obtaining obtaining biobricks in pSB3K3'''====
-
For régulation system:
+
===='''1 - Extraction of BBa_J04450 from DH5α'''====
-
*1.For those transformation products(of yesterday), RBS+LacS+terminator in PSB1C3 plasmid, the liquid culture has been performed for further experiments. The oligopetides for PCR amplification was made bioinformaticly. The transformation for the terminator BBa_B0010 was still fail.
+
-
*2. The plasmid which contains fnr+RBS+LacZ+terminator and fnr+RBS+AmilCP+terminator was extracted. The restiction digest was performeed for them.
+
-
For sensor system:
+
-
*3. The selection of bacterian colonies was achieved by analyzing PCR product. The baterian selected are BphR1 c5 and c6; BphR2 c3 and c4; BphA1 c5 and c6,c7,c8.
+
-
=='''Lab work'''==
+
Sheng
-
A.aero/anaerobic regulation system:
+
{|
-
*BioBrick RBS+LacZ+terminator in plasmid PSB1C3
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
Mini and maxi preparation of 07/10/13 works. We will extract DNA.
 +
|}
-
<u>Transformation for BBa_I732019 terminator</u>
+
Protocol : [[Team:Paris_Saclay/extraction|Low copy plamid extraction]]
-
<p>After ong night culture, we observed 2 tiny colonies on the medium. We continued the experiments by performing another liquid culture at 37°C with ampicillin.</p>
+
-
<p>Transformation for BBa_B0010 was always no go</p>
+
===='''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
-
<u>Verification the transformation of BBa_I004450 in PSB3K3 by digestion and eletrophoresis</u>
+
* SacII :
 +
** DNA : 3µL
 +
** Buffer Blue : 3µL
 +
** SacII : 1µL
 +
** H2O : 21µL
-
<p>We performed 2 types of digestion:</p>
+
* EcoRI/PstI :  
-
<p align="left">Simple digestion:
+
** DNA : 5µL
-
*DNA: 3µl
+
** Buffer Orange : 3µL
-
*Buffe:r 3 µl
+
** EcoRI : 1µL
-
*Enzyme: 1µl
+
** PstI : 1µL
-
*H2O: 23µl
+
** H2O : 20µL
-
*Total: 30µl</p>
+
-
<p>Double digestion:
+
* XhoI/SacII :  
-
*DNA: 5µl
+
** DNA : 5µL
-
*Buffer: 3 µl
+
** Buffer Green : 3µL
-
*Enzyme: 2µl
+
** XhoI : 1µL
-
*H2O: 20µl
+
** SacII : 1µL
-
*Total: 30µl</p>
+
** H2O : 20µL
-
<p>Buffer used:
+
===='''3 - Electrophoresis of the digestion of BBa_J04450'''====
-
*Ecor I+ PST I -> orange
+
-
*Xho I -> green
+
-
*Sac II -> blue
+
-
*XhoI+Sac II -> green</p>
+
-
<p>After the digestion, we performed a eletrophoresis for verification:</p>
+
Zhou
-
{| align="center"
+
{|
-
| style="width:350px;border:1px solid black;" | [[File:PSPCR110713d.jpg|center|350px]]
+
| style="width:350px;border:1px solid black;" |[[[[File:Psgel11107.jpg|500px]]]]
-
| style="width:350px;border:1px solid black;" |
+
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Well 1,7 :XhoI+Sac II
+
* Well 1 : 20µL of pSB3K3 digested by XhoI/SacII+4µL of  6X loading dye
-
*Well 2,8 :Sac II
+
* Well 2 : 20µL of pSB3K3 digested by SacII+4µL of  6X loading dye
-
*Well 3,9 :Ecor I+ PST I
+
* Well 3 : 20µL of pSB3K3 digested by EcoRI/PstI+4µL of  6X loading dye
-
*Well 4,10 :Xho I
+
* Well 4 : 20µL of pSB3K3 digested by XhoI+4µL of  6X loading dye
-
*Well 5,11 : control no digested
+
* Well 5 : 2µL of pSB3K3+1µL of 6X loading dye
-
*gel 0.8%
+
* Well 6 : 6µL of DNA Ladder
-
|}<br>
+
* 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
-
<p>Estimed size and observed size:</p>
+
{|
-
{|border="1" align="center"
+
| 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.
-
|enzyme
+
-
|estimed size
+
-
|observed size
+
-
|-
+
-
|Ecor I+PST I
+
-
|1069bp and 2750 bp
+
-
|1000bp and 2700bp
+
-
|-
+
-
| Xho I
+
-
|2976bp+842bp
+
-
|4000bp
+
-
|-
+
-
|Sac II
+
-
|3819bp
+
-
|4000bp
+
-
|-
+
-
|Xho I+Sac II
+
-
|843bp,616bp,2367bp
+
-
|1200bp and 2000bp
+
|}
|}
-
<p>We confimed the existence of BBa_I04550 in plasmid PSB3K3.</p>
 
-
<br>
 
-
B.PCBs sensor system:
 
-
*Construction for BioBrick promoter promoter BphR1, BphR2, BphA1
 
-
<br>
 
-
<u>Colony PCR</u>
 
-
<p>From the culture of cloning for promoter BphR1, BphR2, BphA1 which we seeded yesterday, we had chosen 8 colonies in total for further test. And like we did for promoter fnr, we used 8 primers for PCR amplification, they were: BphR1 up/down, BphR2 up/down, BphA1 up/down and  Vf/VR.</p>
 
-
<p>In order to make clear this large number of PCR tubes, we classified them into 4 lots. they were:
 
-
<br>
 
-
Mix A : promoter BphR1
 
-
*buffer go ta:(1X) : 5µl
+
==='''B - PBC sensor system'''===
-
*MgCL2: 2µl
+
-
*dNTP: 1µl
+
-
*primers(R1_up/VR or VF/R1_down): 0.125µl
+
-
*DNA: 2µl
+
-
*Enzyme: 0.25µl
+
-
*H2O: about 14.5µl
+
-
*total: 25µl
+
-
<br>
+
-
Mix B : promoter BphR2
+
===='''Objective : obtaining BBa_K1155001, BBa_K1155002, BphR2'''====
-
*buffer go ta:(1X) : 5µl
+
===='''1 - Colony PCR of BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3 in DH5α to check good insertions of BphR1, BphA1, BphR2 in pSB1C3'''====
-
*MgCL2: 2µl
+
-
*dNTP: 1µl
+
-
*primers(R2_up/VR or VF/R2_down): 0.125µl
+
-
*DNA: 2µl
+
-
*Enzyme: 0.25µl
+
-
*H2O: about 14.5µl
+
-
*total: 25µl
+
-
<br>
+
-
Mix C : promoter BphA1
+
Abdou, Anaïs, Zhou
-
*buffer go ta:(1X) : 5µl
+
{|
-
*MgCL2: 2µl
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
*dNTP: 1µl
+
Transformation of BBa_K1155001, BBa_K1155002 and BphR2 protein in DH5α of 07/10/13 work. We will do a Colony PCR.
-
*primers(A1_up/VR or VF/A1_down): 0.125µl
+
|}
-
*DNA: 2µl
+
-
*Enzyme: 0.25µl
+
-
*H2O: about 14.5µl
+
-
*total: 25µl
+
-
<br>
+
-
Mix D :
+
We mix our colonies in 10µL of H2O.
-
*buffer go ta:(1X) : 5µl
+
Used quantities :
-
*MgCL2: 2µl
+
* DNA : 2µL
-
*dNTP: 1µl
+
 
-
*primers(VF/VR): 0.125µl
+
* Mix A : (it was divided in 8tubes for 8 colonies different)
-
*DNA: 2µl
+
** Buffer Go Taq : 50µL
-
*Enzyme: 0.25µl
+
** MgCl2 : 20µL
-
*H2O: about 14.5µl
+
** dNTP : 10µL
-
*total: 25µl
+
** BphR1_Up/VR : 1.25µL for each oligo
-
<br>
+
** 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 :
-
PCR program:
 
[[File:PSPCR1107p.jpg|align="center"|400px]]
[[File:PSPCR1107p.jpg|align="center"|400px]]
-
<br>
 
-
Result : can not find images
+
===='''2 - Electrophoresis of Colony PCR products : BBa_K1155001, BBa_K1155002 and BphR2 in pSB1C3'''====
 +
Abdou, Anaïs
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel21107.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel31107.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel41107.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel51107.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* 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
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
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.
 +
|}
-
<br>
 
{| border="1" align="center"
{| border="1" align="center"
|[[Team:Paris Saclay/Notebook/July/10|<big>Previous day</big>]]
|[[Team:Paris Saclay/Notebook/July/10|<big>Previous day</big>]]

Latest revision as of 23:58, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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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