Team:Paris Saclay/Notebook/July/8

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

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

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
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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.