Team:Paris Saclay/Notebook/August/26
Contents
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
eigem.parissaclay AT gmail DOT com
t@iGEMParisSaclay
aUniversité Paris Sud
a91405 Orsay, France
Copyright © 2015 iGEM Paris Saclay. *This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.