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

(→‎Wednesday 8th July)
(→‎Wednesday 8th July)
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** 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
Line 44: Line 46:
  
 
====Electrophoresis====
 
====Electrophoresis====
−
''by Johan, Coralie, Seong Koo, Pauline''
+
''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  
  
 +
Biobricks:
 
*K115017
 
*K115017
 
*I13504
 
*I13504
 +
 +
We cut our bands with a scalpel
 +
 +
 +
====Purification from agarose gel====
 +
'' by Audrey and Pauline''
 +
 +
With the Nucleospin Gel Clean Up kit from Macherey-Nagel
 +
 +
Biobricks:
 +
* K115017
 +
* I13504
 +
 +
 +
====Plasmid extraction====
 +
''by Coralie''
 +
 +
Biobrick: K1493504
 +
 +
With Nucleospin Kit
 +
 +
 +
====Digestion====
 +
''by Coralie''
 +
 +
Biobrick: 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
 +
Biobricks:
 +
* Extracted DNA:
 +
** K115017
 +
** I13504
 +
* Digested DNA (by SpeI and PstI):
 +
** J23101
 +
** J23106
 +
* Digested DNA (by Not I):
 +
** K1493504
 +
 +
We can conclude:
 +
* J23101 #1: 10ng/µL
 +
* J23101 #2: 13ng/µL
 +
* I13504 #1 #2 #3: 5ng/µL
 +
* J23106: 15ng/µL
 +
* J23117: 8ng/µL
 +
 +
We can't see anything for K115017
 +
 +
 +
====Purification====
 +
''by Coralie''
 +
 +
Biobricks:
 +
* J23101
 +
* J23106
 +
* J23117
 +
 +
With Nucleospin Kit
  
 
====Ligation====
 
====Ligation====

Revision as of 12:52, 17 July 2015

Wednesday 8th July

Lab Work

Transformation

by Audrey

Biobrick: R0051 (2015) On LB + Ampicillin 100ug/mL

Rehydratation

by Seong Koo, Johan

R0051 from different years (2013 and 2014)

Digestion

by Pauline, Coralie

First Digestion:

  • Biobricks:
    • J23101
    • J23106
    • J23117
  • Mix:
    • 10µL of our plasmid with promotor
    • 1µL SpeI
    • 1µL PstI
    • 2µL buffer 10x FastDigest
    • 6µL H2O

Second Digestion:

  • Biobricks:
    • K115017
    • 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

Biobricks:

  • K115017
  • I13504

We cut our bands with a scalpel


Purification from agarose gel

by Audrey and Pauline

With the Nucleospin Gel Clean Up kit from Macherey-Nagel

Biobricks:

  • K115017
  • I13504


Plasmid extraction

by Coralie

Biobrick: K1493504

With Nucleospin Kit


Digestion

by Coralie

Biobrick: 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 Biobricks:

  • Extracted DNA:
    • K115017
    • I13504
  • Digested DNA (by SpeI and PstI):
    • J23101
    • J23106
  • Digested DNA (by Not I):
    • K1493504

We can conclude:

  • J23101 #1: 10ng/µL
  • J23101 #2: 13ng/µL
  • I13504 #1 #2 #3: 5ng/µL
  • J23106: 15ng/µL
  • J23117: 8ng/µL

We can't see anything for K115017


Purification

by Coralie

Biobricks:

  • J23101
  • J23106
  • J23117

With Nucleospin Kit

Ligation

by Coralie, Audrey

  • J23117 + I13504
    • 2,5µL J23117
    • 15µL I13504
    • 1µL T4 DNA Ligase
    • 2µL Buffer T4 DNA Ligase 10x
  • J23106 + I13504
    • 1,5µL J23106
    • 15,5µL I13504
    • 1µL TA DNA Ligase
    • 2µL Buffer T4 DNA Ligase 10x
  • J23201 + 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