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''
  
−
Biobrick: R0051 (2015)
+
* 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 (2013 and 2014)
+
* BBa_R0051 from different years (2013 and 2014)
  
−
====Digestion====
+
===Digestion===
 
''by Pauline, Coralie''
 
''by Pauline, Coralie''
  
−
First Digestion:
+
'''First Digestion:'''
−
* Biobricks:
+
* BBa_J23101
−
** J23101
+
* BBa_J23106
−
** J23106
+
* BBa_J23117
−
** J23117
+
  
−
* Mix:
+
Mix:
−
** 10µL of our plasmid with promotor
+
* 10µL of our plasmid with promotor
−
** 1µL SpeI
+
* 1µL SpeI
−
** 1µL PstI
+
* 1µL PstI
−
** 2µL buffer 10x FastDigest
+
* 2µL buffer 10x FastDigest
−
** 6µL H2O
+
* 6µL H2O
  
−
Second Digestion:
+
'''Second Digestion:'''
−
* Biobricks:
+
* BBa_K115017
−
**K115017
+
* BBa_I13504
−
**I13504
+
  
−
* Mix:
+
Mix:
−
** 10µL of our plasmid with gene
+
* 10µL of our plasmid with gene
−
** 1µL XbaI
+
* 1µL XbaI
−
** 1µL PstI
+
* 1µL PstI
−
** 2µL buffer 10x FastDigest
+
* 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
 
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
 +
[[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
  
−
====Electrophoresis====
+
===Quantification by electrophoresis===
−
''by Johan, Coralie, Seong Koo, Pauline''
+
''by 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  
 +
* Extracted DNA:
 +
** BBa_K115017
 +
** BBa_I13504
 +
* Digested DNA (by SpeI and PstI):
 +
** BBa_J23101
 +
** BBa_J23106
 +
* Digested DNA (by Not I):
 +
** BBa_K1493504
  
−
*K115017
+
[[File:ParisSaclay_08.07.2015_-_Quantification.tif|300px|center]]
−
*I13504
+
<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>
  
−
====Ligation====
+
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''
  
−
* J23117 + I13504
+
* BBa_J23117 + BBa_I13504
−
**2,5µL J23117
+
** 2,5µL J23117
−
**15µL I13504
+
** 15µL I13504
−
**1µL T4 DNA Ligase
+
** 1µL T4 DNA Ligase
−
**2µL Buffer T4 DNA Ligase 10x
+
** 2µL Buffer T4 DNA Ligase 10x
  
−
* J23106 + I13504
+
* BBa_J23106 + BBa_I13504
−
**1,5µL J23106
+
** 1,5µL J23106
−
**15,5µL I13504
+
** 15,5µL I13504
−
**1µL TA DNA Ligase
+
** 1µL TA DNA Ligase
−
**2µL Buffer T4 DNA Ligase 10x
+
** 2µL Buffer T4 DNA Ligase 10x
  
−
* J23201 + I13504
+
* BBa_J23201 + BBa_I13504
 
** 2µL J23101
 
** 2µL J23101
 
** 14,5µL I13504
 
** 14,5µL I13504
Line 78: Line 146:
 
* 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.