Team:Groningen/Notebook/31 July 2009

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Revision as of 10:36, 31 July 2009 by JolandaWitteveen (Talk | contribs)

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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


- Ligation
2 uL Ligase buffer 16 1h 65 10 min
1 ul T4 Ligase 16 1h 65 10 min
11 uL GlpF digested with EcoRI and SpeI 42 2 min 16 1h 65 10 min
11 uL psB1AC3 digested with EcoRi and SpeI 42 2min 16 1h 65 10 min

Metal Accumulation

ArsR-MBP fusion

  • colony PCR MBP:
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 Forever
  • Overnight culture MBP

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).


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