Team:ETH Zurich/Biology/Cloning
From 2011.igem.org
(Difference between revisions)
(→Ligation) |
(→Mediums) |
||
Line 102: | Line 102: | ||
*20 mM MgSO<sub>4</sub> | *20 mM MgSO<sub>4</sub> | ||
adjust to pH 7.5 by adding 1M NaOH | adjust to pH 7.5 by adding 1M NaOH | ||
+ | |||
+ | '''SOC''' | ||
+ | * SOB | ||
+ | * 20 mM glucose | ||
'''LB medium''' | '''LB medium''' | ||
Line 125: | Line 129: | ||
* 1 M CaCl<sub>2</sub> | * 1 M CaCl<sub>2</sub> | ||
* 1 % (w/v) thiamine solution | * 1 % (w/v) thiamine solution | ||
- | * 20 % (w/v) glucose | + | * 20 % (w/v) glucose |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
== '''Used parts from registry''' == | == '''Used parts from registry''' == |
Revision as of 12:54, 19 September 2011
![Menu image preload](/wiki/images/e/e2/ETHZ-bg-overview.png)
![Menu image preload](/wiki/images/0/03/ETHZ-bg-biology.png)
![Menu image preload](/wiki/images/a/ac/ETHZ-bg-process.png)
![Menu image preload](/wiki/images/b/b8/ETHZ-bg-modeling.png)
![Menu image preload](/wiki/images/3/3d/ETHZ-bg-achievements.png)
![Menu image preload](/wiki/images/e/ed/ETHZ-bg-team.png)
Material and methods |
| |||
All protocols, plasmids and primers can be found here. |
Protocols
Double digest for cloning
Variant 1 | Variant 2 | |
Plasmid |
|
|
Insert |
|
|
Ligation
- Ligation mix
- 1:5 ratio of plasmid backbone : Insert
- 1 µl 10 x T4 Ligase buffer (vortex)
- 0.5 µl T4 Ligase
- adjust to 10 µl with ddH2O
- Let the mixture stay for 1h at room temperature
- Denature the ligase at 65 °C for 5 min
Transformation
- Thaw competent cells on ice.
- Add 200 µl cells to 10µl liagtion mix
- Place the mixture put them on ice for 30 min
- Heatschock 30 sec at 42 °C
- Place the mixture again on ice for 2 min
- Add 4 x SOC medium (800 µl)
- Let them growth for 60 min at 37°C
- Spin cells down for 5 min at low speed
- Remove the supernatant, resuspend the cells
- Spread 100 µl of the cells onto the plates.
PCR
Colonie PCR
Whole plasmid PCR
Mediums
SOB
- 0.5 % (w/v) yeast extract
- 2 % (w/v) tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
adjust to pH 7.5 by adding 1M NaOH
SOC
- SOB
- 20 mM glucose
LB medium
- 10 g Bacto-tryptone
- 5 g yeast extract
- 10 g NaCl
Total 1 l
M9 minimal medium
10x M9
- 12.8 g Na2HPO4
- 3 g KH2PO4
- 0.5 g NaCl
- 1 g NH4Cl
Total 100 ml
autoclave seperatly
- 10 x M9
- 1 M MgSO4
- 1 M CaCl2
- 1 % (w/v) thiamine solution
- 20 % (w/v) glucose
Used parts from registry
- BBa R0040 PTet
- BBa R0051 λP
- BBa R0010 Plac
- BBa R0061 Plux
- BBa J23100 Pconst
- BBa C0061 LuxILVA
- BBa C0040 TetRLVA
- BBa C0051 CI
- BBa C0062 LuxR
- BBa B0015 Double Terminator
Plasmid list
Primer list
Synthesized parts
- LacIM1
- AlcR (codon optimized)