Team:Paris Saclay/Notebook/August/5

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Obtaining RBS-AmilCP-Term'''====
+
===='''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
Nadia, XiaoJing
-
Digestion
+
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
{|
{|
-
| style="width:350px;border:1px solid black;" | IMAGE
+
| style="width:350px;border:1px solid black;" |[[File:Psgel10508.jpg| 400px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Well 1 : 6µL DNA Ladder
+
* Well 1 : 6µL of DNA Ladder
-
*Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
+
* Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI + 6µl of 6X loading dye
-
*Gel : 0.8%
+
* 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
 +
 
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will sequence it.
|}
|}
-
Expected sizes :
+
===='''Objective : obtaining BBa_K1155007'''====
-
*PSB3K3 : 2750kb
+
===='''1 - Digestion of BBa_I732017 by EcoRI/SpeI'''====
-
*GFP : 1069kb
+
-
We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
+
Abdou, Damir, Nadia,
-
=====2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1=====
+
Used quantities :
 +
* BBa_I732017 : 41µL
 +
* Buffer FD : 5µL
 +
* EcoRI : 2µL
 +
* SpeI : 2µL
-
''2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1''
+
We incubate the digestion  at 37°C for 1h30.
-
Anaïs
+
===='''2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI'''====
 +
 
 +
Damir, Nadia
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_eelution.jpg|350px]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel20508.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
*Well 1 : 6µL DNA Ladder
+
* Well 1 : 6µL of DNA Ladder
-
*Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
+
* Well 2 : 40µL of BBa_I732017 digested by EcoRI/Spe I+ 8µL 6X loading dye
-
*Gel : 0.8%
+
* Gel : 1.5%
|}
|}
-
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
+
Expected sizes :
 +
* RBS-LacZ : 3093 bp
 +
* pSB1A2 : 2079 bp
-
''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will make an electro-elution to extract RBS-LacZ.
 +
|}
-
Nadia
+
===='''3 - PCR of pSB1C3'''====
-
Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
+
Nadia, XiaoJing
-
We let the plasmid precipitate during the night.
+
Used quantities :
 +
* pSB1C3 : 2µL
 +
* Buffer phusion : 10µL
 +
* Oligo 64 : 2.5µL
 +
* Oligo 65 : 2.5µL
 +
* dNTP : 1µL
 +
* enzyme Phusion : 0.25µL
 +
* H2O : 36.75µL
-
===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
+
PCR program :
-
=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
+
Hybridation temperature gradient :
-
Anaïs
+
-
*Colony counting :
+
A - 65°C/
-
**Low concentration petri dish : 47 colonies
+
B - 64.7°C/
-
**High concentration petri dish : 145 colonies
+
C - 64.1°C/
 +
D - 63.1°C/
 +
E - 62°C/
 +
F - 61.2°C/
 +
G - 60.5°C/
 +
H - 60°C/
-
*Picking of 25 colonies
+
[[File:PsPCRC30508.jpg|400px]]
-
*Preparation of 700µL of Master mix
+
===='''3 - Electrophoresis of PCR of pSB1C3'''====
-
**H<sub>2</sub>O : 590µL
+
-
**dNTP : 28µL
+
-
**VF2 primer : 3.5µL
+
-
**VR primer : 3.5µL
+
-
**DreamTaq buffer 10x : 70µL
+
-
**DreamTaq enzyme : 5µL
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
+
Nadia, XiaoJing
-
PCR Program :
+
{|
 +
| 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%
 +
|}
-
[[File:PsPcr808.jpg|400px]]
+
Expected sizes :  
 +
* pSB1C3 : 2070bp
-
=====2 - Gel electrophoresis of the colony PCR products=====
+
{|
-
Anaïs, Damir
+
| 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
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
+
Transformation of 07/31/13 works. We will do a PCR Colony.
-
*6µL DNA Ladder
+
-
*10µL sample per well
+
-
*Gel : 0.8%
+
|}
|}
-
Expected size : 3583bp
+
We took a single colony and resuspend in 10µL H2O. For each Biobrick, we did 25 PCR Colonies.
-
Colonies 10, 14, 15 exhibit plasmids with the right length.
+
Used quantities :
-
====3 - PCR product (made the 08/01/2013) purification====
+
PCR preparation mix for 25 different colonies including:
-
Damir
+
** Oligo 44 : 3.5µL
 +
** Oligo 45 : 3.5µL
 +
** Buffer Dream Taq : 70µL
 +
** dNTP : 28µL
 +
** Dream Taq : 5µL
 +
** H2O : 590µL 
-
available quantity:
+
PCR reaction:
-
* FNR Part1 : 10 µl
+
* DNA : 2µL of resuspend colony
-
* FNR Part2 : 19 µl
+
* Mix PCR  :23µL
-
* RBS FNR Part1 :16.1µl
+
Total volume: 25µL
-
* RBS BphR2 Part1 : 28µl
+
-
* BphR2 Part1 : 16.4 µl
+
-
* BphR2 Part2 : 18.9 µl
+
-
Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
+
PCR Program :  
-
<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
+
[[File:PsPCR0508.jpg|400px]]
-
 
 +
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 +
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 +
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 +
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 +
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Latest revision as of 01:25, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

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

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

Psgel10508.jpg
  • 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

Psgel20508.jpg
  • 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/

PsPCRC30508.jpg

3 - Electrophoresis of PCR of pSB1C3

Nadia, XiaoJing

Psgel30508.jpg
  • 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 :

PsPCR0508.jpg


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