"
Team:Paris Saclay/Notebook/August/8
From 2013.igem.org
(Difference between revisions)
| Line 54: | Line 54: | ||
=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies===== | =====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies===== | ||
| - | |||
Anaïs | Anaïs | ||
| Line 60: | Line 59: | ||
**Low concentration petri dish : 47 colonies | **Low concentration petri dish : 47 colonies | ||
**High concentration petri dish : 145 colonies | **High concentration petri dish : 145 colonies | ||
| + | |||
| + | *Picking of 25 colonies | ||
*Preparation of 700µL of Master mix | *Preparation of 700µL of Master mix | ||
| Line 69: | Line 70: | ||
**DreamTaq enzyme : 5µL | **DreamTaq enzyme : 5µL | ||
| + | Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]] | ||
| + | |||
| + | PCR Program : | ||
| + | [IMAGE] | ||
| + | |||
| + | =====2 - Gel electrophoresis of the colony PCR products===== | ||
| + | Anaïs, Damir | ||
| + | |||
| + | {| | ||
| + | | style="width:350px;border:1px solid black;" | IMAGE | ||
| + | | style="width:350px;border:1px solid black;" | | ||
| + | *6µL DNA Ladder | ||
| + | *10µL sample per well | ||
| + | *Gel : 0.8% | ||
| + | |} | ||
{{Team:Paris_Saclay/incl_fin}} | {{Team:Paris_Saclay/incl_fin}} | ||
Revision as of 15:51, 9 August 2013
Contents |
Notebook : August 8
Lab work
A - Aerobic/Anaerobic regulation system
Obtaining the PSB3K3 backbone plasmid
1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion was right
Damir, Nadia
| IMAGE |
|
Expected sizes :
- PSB3K3 : 2750kb
- GFP : 1069kb
We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1
2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1
Anaïs
| IMAGE |
|
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel
Nadia
Protocol : Electroelution
We let the plasmid precipitate during the night.
Obtaining RBS_LacZ+Term_PSB1C3
1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies
Anaïs
- Colony counting :
- Low concentration petri dish : 47 colonies
- High concentration petri dish : 145 colonies
- Picking of 25 colonies
- Preparation of 700µL of Master mix
- H2O : 590µL
- dNTP : 28µL
- VF2 primer : 3.5µL
- VR primer : 3.5µL
- DreamTaq buffer 10x : 70µL
- DreamTaq enzyme : 5µL
Protocol : Colony PCR
PCR Program : [IMAGE]
2 - Gel electrophoresis of the colony PCR products
Anaïs, Damir
| IMAGE |
|
