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Tuesday 25th August

Lab Work

Electrophoresis purification

by Audrey

• BBa_K1707004 #5 (digested by SpeI and EcoRI)
• BBa_K1707022 #1 (digested by XbaI and PstI)

Agarose gel 1%, migration 90V

Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#5, 3. BBa_K1707022#1, 4. Empty, 5. Empty

We cut corresponding bands with a scalpel.

Purification

by Audrey

• BBa_K1707022 #1
• BBa_K1707023 #1
• BBa_K1707004 #5
• BBa_R0040 #1

With Macherey Nagel purification kit

Quantification

by Audrey

Agarose gel 1%, migration 90V. For each sample:

• 5 µL plasmid
• 5 µL H2O
• 2 µL Ladder 6x

Quantification, from left to right: 1. DNA Ladder, 2. BBa_R0040 (EcoRI+XbaI), 3. BBa_K1707004#5 (SpeI+EcoRI), 4. BBa_K1707004#5 (SpeI+PstI), 5. BBa_K1707022, 6. BBa_K1707023, 7. Empty, 8. Empty, 9. Empty, 10. Empty

We can conclude:

• BBa_K1707022 #1: nothing can be seen
• BBa_K1707023 #1: nothing can be seen
• BBa_K1707004 #5 (SpeI + EcoRI): 20 µg/µL
• BBa_K1707004 #5 (SpeI + PstI): 75 µg/µL
• BBa_R0040 #1: 75 µg/µL

PCR for the Gibson experiment

by Audrey

Amplification Thermometer RNA BBa_K115017

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion
• 1 μL dNTP 10mM
• 0,5 μL Forward primer
• 0,5 μL Reverse primer
• 1μL plasmid BBa_K115017 v10
• 0,25 μL DNA Pol Phusion

PCR Cycle:

• 98°C - 30 seconds
• 30 cycles:
  • 98°C - 5 seconds
  • 69,8°C - 30 seconds
  • 72°C - 10 seconds
• 72°C - 10 minutes
• 4°C for ever

Amplification (reverse PCR)

BioBricks with cI and cI857:

• BBa_K1707013
• BBa_K1707019
• BBa_K1707020
• BBa_K1707035
• BBa_K1707036

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion
• 1 μL dNTP 10mM
• 0,5 μL Forward primer
• 0,5 μL Reverse primer
• 1μL plasmid (dilution 1/10 or 1/100)
• 0,25 μL DNA Pol Phusion

PCR Cycle:

• 98°C - 30 seconds
• 30 cycles:
  • 98°C - 5 seconds
  • 65,6°C - 30 seconds
  • 72°C - 3 minutes
• 72°C - 10 minutes
• 4°C for ever

Amplification (reverse PCR)

• BBa_K1707021

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion
• 1 μL dNTP 10mM
• 0,5 μL Forward primer
• 0,5 μL Reverse primer
• 1μL plasmid (dilution 1/10 or 1/100)
• 0,25 μL DNA Pol Phusion

PCR Cycle:

• 98°C - 30 seconds
• 30 cycles:
  • 98°C - 5 seconds
  • 65,6°C - 30 seconds
  • 72°C - 3 minutes
• 72°C - 10 minutes
• 4°C for ever

Amplification (reverse PCR)

• BBa_K1707027

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion
• 1 μL dNTP 10mM
• 0,5 μL Forward primer
• 0,5 μL Reverse primer
• 1μL plasmid (dilution 1/10 or 1/100)
• 0,25 μL DNA Pol Phusion

PCR Cycle:

• 98°C - 30 seconds
• 30 cycles:
  • 98°C - 5 seconds
  • 62,6°C - 30 seconds
  • 72°C - 2 minutes
• 72°C - 10 minutes
• 4°C for ever

Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

We load on gel 5μL of PCR product.

Quantification, from left to right: 1. DNA Ladder, 2. BBa_K1707020, 3. BBa_K1707030, 4. BBa_I13602#1, 5. BBa_I13602#2, 6. Empty, 7. Empty, 8. Empty, 9. Empty, 10. Empty

We can conclude that we need to start over all PCR except BBa_K115017 which seems to be OK.

Member present:

• Instructors: Claire
• Students: Audrey

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