From 2009.igem.org
Wet
GVP Cluster
Transporters
GlpF
- Restiction/ligase and transformation of GlpF in psB1AC3
Restiction
10ul
| GlpF PCR
|
2 ul
| Buffer
|
05.ul
| EcoRI
|
0.5 ul
| SpeI
|
7 uL
| MQ
|
- - 37 30min
- -Inactivation on Gel
- -Extraction from gel
2 uL
| Ligase buffer
|
1 ul
| T4 Ligase
|
11 uL
| GlpF digested with EcoRI and SpeI
|
11 uL
| psB1AC3 digested with EcoRi and SpeI
|
Metal Accumulation
ArsR-MBP fusion
-
colony PCR MBP
Component | amount
|
2x Phusion MM
| 12.5 uL
|
MQ
| 9.5 uL
|
MBP Fw
| 1 uL
|
MBP Rev
| 1 uL
|
Colony MBP
| 1 uL
|
|
|
Touchdown MBP program
| Temperature
| Time
|
Denaturing
| 98°
| 2.00 min
|
| Touchdown 10X 60->50
|
Denaturing
| 98°
| 30 sec
|
Annealing
| 60°->50°
| 30 sec
|
Elongation
| 72°
| 30 sec
|
| End cycles
|
Start Cycles 25X
|
Denaturing
| 98°
| 30sec
|
Annealing
| 60°
| 30 sec
|
Elongation
| 72°
| 30 sec
|
| End cycles
|
Final elongation
| 72°
| 7 min
|
Hold
| 4°
| Forever
|
|
|
-
Vectors
Dry
We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was previously measured using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR might qualify as such).