Difference between revisions of "Team:Paris Saclay/Notebook/July/8"

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{{Team:Paris_Saclay/notebook_header}}
 
=Wednesday 8th July=
 
=Wednesday 8th July=
 
==Lab Work==
 
==Lab Work==
−
====Transformation====
+
===Transformation===
 
''by Audrey''
 
''by Audrey''
  
−
R0051
+
* BBa_R0051 (2015)
 
On LB + Ampicillin 100ug/mL  
 
On LB + Ampicillin 100ug/mL  
  
−
====Rehydratation====
+
===Rehydratation===
 
''by Seong Koo, Johan''
 
''by Seong Koo, Johan''
  
−
R0051 from different years
+
* BBa_R0051 from different years (2013 and 2014)
  
−
====Digestion====
+
===Digestion===
 
''by Pauline, Coralie''
 
''by Pauline, Coralie''
  
−
10uL of our plasmid with promotor
+
'''First Digestion:'''
−
1uL SpeI
+
* BBa_J23101
−
1uL PstI
+
* BBa_J23106
−
2uL buffer 10x FD
+
* BBa_J23117
  
−
*J23101
+
Mix:
−
*J23106
+
* 10µL of our plasmid with promotor
−
*J23117
+
* 1µL SpeI
 +
* 1µL PstI
 +
* 2µL buffer 10x FastDigest
 +
* 6µL H2O
  
−
10uL of our plasmid with gene
+
'''Second Digestion:'''
−
1uL XbaI
+
* BBa_K115017
−
1uL PstI
+
* BBa_I13504
−
2uL buffer 10x FD
+
−
After this step, we separate the sequence we need from the sequence we doesn't with electrophoresis
+
  
−
*K115017
+
Mix:
−
*I13504
+
* 10µL of our plasmid with gene
 +
* 1µL XbaI
 +
* 1µL PstI
 +
* 2µL buffer 10x FastDigest
  
−
====Electrophoresis====
+
Incubation 1h30, 37°C
−
''by Johan, Coralie, Seong Koo, Pauline''
+
 
 +
After this step, we separate the sequence we need from the sequence we don't with electrophoresis
 +
 
 +
===Electrophoresis===
 +
''by Coralie, Pauline''
  
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5uL of BET
 
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5uL of BET
 
Migration 0,06A 80V  
 
Migration 0,06A 80V  
  
−
*K115017
+
* BBa_K115017
−
*I13504
+
* BBa_I13504
  
−
====Ligation====
+
We cut our bands with a scalpel
 +
[[File:ParisSaclay 080715 vérif découpe.tif|200px|center]]
 +
<html><i><p>Cut verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K115017, 3. BBa_I13504#1, 4. BBa_I13504#2, 5. BBa_I13504#3, 6. Empty</i></p></i></html>
 +
 
 +
===Purification from agarose gel===
 +
'' by Audrey and Pauline''
 +
 
 +
With the Nucleospin Gel Clean Up kit from Macherey-Nagel
 +
 
 +
* BBa_K115017
 +
* BBa_I13504
 +
 
 +
 
 +
===Plasmid extraction===
 +
''by Coralie''
 +
 
 +
* BBa_K1493504
 +
 
 +
With Nucleospin Kit
 +
 
 +
===Digestion===
 +
''by Coralie''
 +
 
 +
* BBa_K1493504
 +
 
 +
Mix (2tubes):
 +
* 33µL H2O
 +
* 4µL Buffer FastDigest 10x
 +
* 1µL NotI
 +
 
 +
We use 2µL of plasmid in each tube
 +
 
 +
Incubation 1h30, 37°C
 +
 
 +
===Quantification by electrophoresis===
 +
''by Pauline''
 +
 
 +
Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5uL of BET
 +
Migration 0,06A 80V
 +
* Extracted DNA:
 +
** BBa_K115017
 +
** BBa_I13504
 +
* Digested DNA (by SpeI and PstI):
 +
** BBa_J23101
 +
** BBa_J23106
 +
* Digested DNA (by Not I):
 +
** BBa_K1493504
 +
 
 +
[[File:ParisSaclay_08.07.2015_-_Quantification.tif|300px|center]]
 +
<html><p><i>Wells 1-2: Verification/ Wells 4-11: Quantification; from left to right: 1. BBa_K1493504#1, 2. BBa_K1493504#2, 3. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 4. BBa_J23101#1, 5. BBa_J23101#2, 6. BBa_K115017, 7. BBa_I13504#1, 8. BBa_I13504#2, 9. BBa_I13504#3, 10. BBa_J23106, 11. BBa_J23117, 12. Empty</i></p></html>
 +
 
 +
We can conclude:
 +
* BBa_J23101 #1: 10ng/µL
 +
* BBa_J23101 #2: 13ng/µL
 +
* BBa_I13504 #1 #2 #3: 5ng/µL
 +
* BBa_J23106: 15ng/µL
 +
* BBa_J23117: 8ng/µL
 +
 
 +
We can't see anything for BBa_K115017
 +
 
 +
===Purification===
 +
''by Coralie''
 +
 
 +
* BBa_J23101
 +
* BBa_J23106
 +
* BBa_J23117
 +
 
 +
With Nucleospin Kit
 +
 
 +
===Ligation===
 
''by Coralie, Audrey''
 
''by Coralie, Audrey''
  
−
2,5uL J23117
+
* BBa_J23117 + BBa_I13504
−
15uL I13504
+
** 2,5µL J23117
−
1uL Ligase
+
** 15µL I13504
−
2uL buffer 10x
+
** 1µL T4 DNA Ligase
 +
** 2µL Buffer T4 DNA Ligase 10x
  
−
1,5uL J23106
+
* BBa_J23106 + BBa_I13504
−
15,5uL I13504
+
** 1,5µL J23106
−
1uL Ligase
+
** 15,5µL I13504
−
2uL buffer 10x
+
** 1µL TA DNA Ligase
 +
** 2µL Buffer T4 DNA Ligase 10x
  
−
2uL J23101
+
* BBa_J23201 + BBa_I13504
−
14,5uL I13504
+
** 2µL J23101
−
1uL Ligase
+
** 14,5µL I13504
−
2uL buffer 10x
+
** 1µL Ligase
 +
** 2µL buffer 10x
  
−
Waiting all the night under 16°C
+
Incubation ON, 16°C
  
 
'''Members present:'''
 
'''Members present:'''
 
* Instructors and advisors: Alice.
 
* Instructors and advisors: Alice.
 
* Students: Johan, Seong Koo, Audrey, Coralie, Pauline
 
* Students: Johan, Seong Koo, Audrey, Coralie, Pauline
 +
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 21:13, 18 September 2015

Wednesday 8th July

Lab Work

Transformation

by Audrey

  • BBa_R0051 (2015)

On LB + Ampicillin 100ug/mL

Rehydratation

by Seong Koo, Johan

  • BBa_R0051 from different years (2013 and 2014)

Digestion

by Pauline, Coralie

First Digestion:

  • BBa_J23101
  • BBa_J23106
  • BBa_J23117

Mix:

  • 10µL of our plasmid with promotor
  • 1µL SpeI
  • 1µL PstI
  • 2µL buffer 10x FastDigest
  • 6µL H2O

Second Digestion:

  • BBa_K115017
  • BBa_I13504

Mix:

  • 10µL of our plasmid with gene
  • 1µL XbaI
  • 1µL PstI
  • 2µL buffer 10x FastDigest

Incubation 1h30, 37°C

After this step, we separate the sequence we need from the sequence we don't with electrophoresis

Electrophoresis

by Coralie, Pauline

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5uL of BET Migration 0,06A 80V

  • BBa_K115017
  • BBa_I13504

We cut our bands with a scalpel

ParisSaclay 080715 vérif découpe.tif

Cut verification, from left to right: 1. DNA Ladder, 2. BBa_K115017, 3. BBa_I13504#1, 4. BBa_I13504#2, 5. BBa_I13504#3, 6. Empty

Purification from agarose gel

by Audrey and Pauline

With the Nucleospin Gel Clean Up kit from Macherey-Nagel

  • BBa_K115017
  • BBa_I13504


Plasmid extraction

by Coralie

  • BBa_K1493504

With Nucleospin Kit

Digestion

by Coralie

  • BBa_K1493504

Mix (2tubes):

  • 33µL H2O
  • 4µL Buffer FastDigest 10x
  • 1µL NotI

We use 2µL of plasmid in each tube

Incubation 1h30, 37°C

Quantification by electrophoresis

by Pauline

Preparation of Agarose Gel 1%, 0,5g in 50mL of 1X TAE, 0,5uL of BET Migration 0,06A 80V

  • Extracted DNA:
    • BBa_K115017
    • BBa_I13504
  • Digested DNA (by SpeI and PstI):
    • BBa_J23101
    • BBa_J23106
  • Digested DNA (by Not I):
    • BBa_K1493504
ParisSaclay 08.07.2015 - Quantification.tif

Wells 1-2: Verification/ Wells 4-11: Quantification; from left to right: 1. BBa_K1493504#1, 2. BBa_K1493504#2, 3. DNA Ladder, 4. BBa_J23101#1, 5. BBa_J23101#2, 6. BBa_K115017, 7. BBa_I13504#1, 8. BBa_I13504#2, 9. BBa_I13504#3, 10. BBa_J23106, 11. BBa_J23117, 12. Empty

We can conclude:

  • BBa_J23101 #1: 10ng/µL
  • BBa_J23101 #2: 13ng/µL
  • BBa_I13504 #1 #2 #3: 5ng/µL
  • BBa_J23106: 15ng/µL
  • BBa_J23117: 8ng/µL

We can't see anything for BBa_K115017

Purification

by Coralie

  • BBa_J23101
  • BBa_J23106
  • BBa_J23117

With Nucleospin Kit

Ligation

by Coralie, Audrey

  • BBa_J23117 + BBa_I13504
    • 2,5µL J23117
    • 15µL I13504
    • 1µL T4 DNA Ligase
    • 2µL Buffer T4 DNA Ligase 10x
  • BBa_J23106 + BBa_I13504
    • 1,5µL J23106
    • 15,5µL I13504
    • 1µL TA DNA Ligase
    • 2µL Buffer T4 DNA Ligase 10x
  • BBa_J23201 + BBa_I13504
    • 2µL J23101
    • 14,5µL I13504
    • 1µL Ligase
    • 2µL buffer 10x

Incubation ON, 16°C

Members present:

  • Instructors and advisors: Alice.
  • Students: Johan, Seong Koo, Audrey, Coralie, Pauline

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Contact
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France Copyright © 2015 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.