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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

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

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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