Team:Bordeaux/NotebookJune23
From 2014.igem.org
19th June
Molecular biology
- PCR of ELP-40 and ELP-60
=> Dilution primer 100µM => 10µM Primer forward: 1µl primer + 9µl H2O (Tm=58°C) Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)
Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C
Mix for 11 tubes - Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)
Digestion plasmid Digestion insert H2O 7,5µl 7,5µl 10X buffer 4 2µl 2µl XbaI 0,25µl 0,25µL EcoRI 0,25µl 0,25µl DNA 10µl 10µl - Digestion vector psB6A1 :
Digestion plasmid H2O 7,5µl 10X buffer 4 2µl XbaI 0,25µl EcoRI 0,25µl DNA 10µl - Digestion psB1C3 and PCR 40 and 60
OD PCR tube 40= 282,5 ng/μl
OD PCR tube 60= 366,3 ng/μl
Tube 40 = 0,88 DNA +9,12µl H2O
Tube 60 = 2,5 DNA + 7,5 µl H2O
H2O | 357,5 µl |
Buffer 5X | 110 µl |
Primer | 27,5 µl |
dNTP | 11µl |
Thermal cycler :
25 cycles
3min 95°C
30sec 95°C
30sec 95°C
25 cycles
30sec at all temperatures tested
3sec 72°C
5min 72°C
3sec 72°C
5min 72°C
With DNA = 6μl vector + 4μl H2O
With DNA= 0.56μl Miniprep product + 9,44μl H2O
Production
We want to do a preculture test to have a better production.