From 2009.igem.org
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| | ==Dry== | | ==Dry== |
| | + | The first part of the day was spent processing the data of yesterday's [[http://www.jbc.org/cgi/content/abstract/M400037200v1 paper]]. However when we calculated the drop in concentration of As(III) outside the membrane vesicle we found that it did not drop significantly enough to make a model of the efflux rate of As(III) depending on concentration of Arsenic in the cell. However we were able to find a paper which a more likely candidate. [[http://www.jbc.org/cgi/content/full/272/1/326?view=long&pmid=8995265 Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.]] |
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| | {{Team:Groningen/Notebook/Day/Footer}} | | {{Team:Groningen/Notebook/Day/Footer}} |
Revision as of 13:30, 24 July 2009
Wet
GVP Cluster
Transporters
Below the PCR1.1 and PCR2 of 24/07, PCR 2 shows a fragment of desired size (~261pb) and is excised for DNA extraction for PCR3. Our previously excised PCR1 fragment will be used as template(stil containing EcoRI)and see if we can reproduce products PCR1 and make PCR1.1.
PCR1.1
| Component | amount
|
| 2x Phusion MM
| 12.5 uL
|
| MQ
| 9.5 uL
|
| F2_L-ATG-mut
| 1 uL
|
| mut1_revcomp
| 1 uL
|
| DNA
| 1 uL
|
|
|
| PCR1.1/PCR2 program
| Temperature
| Time
|
| Denaturing
| 98°
| 2.00 min
|
|
| Touchdown 10X 60->50
|
| Denaturing
| 98°
| 10 sec
|
| Annealing
| 60°->50°
| 10 sec
|
| Elongation
| 72°
| 30 sec
|
|
| End cycles
|
| Start Cycles 30X
|
| Denaturing
| 98°
| 10 sec
|
| Annealing
| 60°
| 10 sec
|
| Elongation
| 72°
| 30 sec
|
|
| End cycles
|
| Final elongation
| 72°
| 5 min
|
| Hold
| 4°
| Forever
|
|
|
PCR2
| Component | amount
|
| 2x Phusion MM
| 12.5 uL
|
| MQ
| 9.5 uL
|
| Rev
| 1 uL
|
| mut2_revcom
| 1 uL
|
| DNA
| 1 uL
|
|
|
|
Metal Accumulation
Vectors
Dry
The first part of the day was spent processing the data of yesterday's [paper]. However when we calculated the drop in concentration of As(III) outside the membrane vesicle we found that it did not drop significantly enough to make a model of the efflux rate of As(III) depending on concentration of Arsenic in the cell. However we were able to find a paper which a more likely candidate. [Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.]
"
