From 2009.igem.org
It's my birthday!!! (Michael)
So cake it is....
Wet
GVP Cluster
- → DONE buy a nice cake and eat it ;)
- → TODO isolate plasmids from overnight precultures
- → DONE perform a ligation between cut vector and GVP fragment
- → DONE transform E.coli TOP10 competent cells with ligation product
Ligation
- 2 uL Ligase buffer
- 1 ul T4 Ligase
- 8 uL plasmid pSB1AC3 digested with PstI and SpeI
- 4 uL insert GVP restricted with XbaI and PstI
Incubate:
- 25°C 30min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 50 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp50 plates
Transporters
GlpF
Restriction, ligation and transformation as noted below.
Metal Accumulation
Restriction
10 ul
| GlpF PCR
|
1.5 ul
| Fast DigestBuffer
|
0.5 ul
| XbaI
|
0.5 ul
| PstI
|
|
|
10 ul
| ArsRMBP fusie (ligatie overnight 04-08-09)
|
1.5 ul
| Fast DigestBuffer
|
0.5 ul
| XbaI
|
0.5 ul
| PstI
|
|
|
11 ul
| psB1AC3 (30/07/09 nienke)
|
5 ul
| Fast DigestBuffer
|
2.5 ul
| XbaI
|
2.5 ul
| PstI
|
|
|
|
- 37 30min
- Inactivation on Gel
- Extraction from gel
Ligation
12 uL
| ArsRMBP
|
5 uL
| psB1AC3
|
2 uL
| ligase
|
1uL
| T4 ligase
|
|
|
|
2 uL
| Ligase buffer
|
| 16° 1h → 65° 10 min
|
1 ul
| T4 Ligase
|
|
11 uL
| GlpF digested with EcoRI and SpeI
| 42° 2 min →
|
11 uL
| psB1AC3 digested with EcoRi and SpeI
|
|
|
- Tranformation
- add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.
Incubate:
- 30 min @ ice
- 5 min 37°
- 5min @ ice
-
Dry