Wednesday 26th August

Lab Work

Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

We load on gel 5μL of PCR product.

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K1707035 (10%), 3. BBa_K1707035 (1%), 4. BBa_K1707036 (10%), 5. BBa_K1707036 (1%), 6. BBa_K1707021 (10%), 7. BBa_K1707021 (1%), 8. BBa_K1707027 (10%), 9. BBa_K1707027 (1%), 10. DNA Ladder, 11. Empty, 12. Empty

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K1707013 (10%), 3. BBa_K1707013 (1%), 4. BBa_K1707019 (10%), 5. BBa_K1707019 (1%), 6. BBa_K1707020 (10%), 7. BBa_K1707020 (1%), 8. BBa_K1707030 (10%), 9. BBa_K1707030 (1%), 10. DNA Ladder

Verification of PCR products, from left to right: 1. DNA Ladder, 2. BBa_K115017, 3. BBa_K115017, 4. BBa_K115017, 5. BBa_K115017

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

PCR on colony

by Audrey

• BBa_K1707037 #1 to #9

PCR mix for each tube:

• 9,75 µL H2O
• 5 µL Buffer Taq 10x
• 1 μL dNTP 10mM
• 0,5 μL Forward primer iPS43
• 0,5 μL Reverse primer iPS44
• 3 μL 25mM MgCl2
• 0,25 μL Taq Pol

PCR Cycle:

• 95°C - 6 + 2 min
• 30 cycles:
  • 95°C - 30 seconds
  • 51,8°C - 30 seconds
  • 72°C - 2 minutes
• 72°C - 10 minutes
• 4°C for ever

Electrophoresis

by Audrey

Agarose gel 1%, migration 90V

PCR Products of BBa_K1707037

Verification of K1707037 colony PCR, from left to right: 1. DNA Ladder, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. #7, 9. #8, 10. #9

We can't conclude anything because we can't see any positive result. So we choose arbitrary 5 clones for different extractions.

Electrophoresis purification

by Audrey

PCR product of BBa_K175017 from the 08/25/2015

In each weel of the agarose gel, we put 45μL of PCR Product + 15 μL Ladder 6x

Agarose gel 1%, Migration 90V

We cut bands with a scalpel and purificate them by the Macherey-Nagel gel purification kit

Plasmid extraction

by Audrey

• BBa_K1707022 #1
• BBa_K1707023 #2
• BBa_K1707037 #5 to #9
• BBa_K1707004 #2 (from two different cultures)

With Macherey-Nagel Extraction kit

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

Agarose gel 1%, migration 90V

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

Reverse PCR

by Audrey

First

• BBa_K1707013
• BBa_K1707019
• BBa_K1707020
• BBa_K1707030
• BBa_K1707035
• BBa_K1707036

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion 5x
• 1 μL dNTP 10mM
• 0,5 μL Forward primer insertion cI
• 0,5 μL Reverse primer RV
• 1μL plasmid
• 0,25 μL DNA Pol Phusion
• With and without DMSO 1,5μL

Second

• BBa_K1707021
• BBa_K1707027

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion 5x
• 1 μL dNTP 10mM
• 0,5 μL Forward primer insertion CFP or GFP
• 0,5 μL Reverse primer insertion RV
• 1μL plasmid
• 0,25 μL DNA Pol Phusion
• With and without DMSO 1,5μL

Control (-) for primers:

PCR mix for each tube:

• 36,75 µL H2O
• 10 µL Buffer Phusion 5x
• 1 μL dNTP 10mM
• 0,5 μL Forward primer insertion CFP/GFP/cI + 0,5μL iPS44 or 0,5 μL Reverse primer insertion RV + 0,5μL iPS43
• 1μL plasmid BBa_K1707013 or BBa_K1707021 or BBa_K1707027
• 0,25 μL DNA Pol Phusion
• With and without DMSO 1,5μL

PCR Cycle:

• 98°C - 30 seconds
• 3 cycles:
  • 98°C - 5 seconds
  • 55°C - 30 seconds
  • 72°C - 3 minutes
• 27 cycles:
  • 98°C - 5 seconds
  • 65°C - 30 seconds
  • 72°C - 3 minutes
• 72°C - 10 minutes
• 4°C for ever

Member present:

• Instructors: Claire
• Students: Audrey

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