Team:HokkaidoU Japan/Notebook/aggregation Week 4
From 2012.igem.org
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Ethanol precipitation for digestion and gel extraction product. | Ethanol precipitation for digestion and gel extraction product. | ||
- | + | #Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol. | |
- | + | #Centrifuged in 15000rpm, 10min at 4C. | |
- | + | #Remove supernatant and added 220ul of 70% ethanol. | |
- | + | #Centrifuged in 15000rpm, 10min at 4C. | |
- | + | #Remove supernatant and air drying in room temperature then added 10ul of DW. | |
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]] | [[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]] |
Revision as of 08:43, 24 July 2012
Contents |
July 23th
Mini-prep
mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.
July 24th
Electrophoresis
Electrophoresis for Ag43(mini-preped above) and Ag43 digestion results(digested with EcoRI and SpeI) [[image:|thumb|Mini-prep result]]
In this digestion result, we knew that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use in digestion.
Gel extraction
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.
Ethanol precipitation
Ethanol precipitation for digestion and gel extraction product.
- Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
- Centrifuged in 15000rpm, 10min at 4C.
- Remove supernatant and added 220ul of 70% ethanol.
- Centrifuged in 15000rpm, 10min at 4C.
- Remove supernatant and air drying in room temperature then added 10ul of DW.
In this result, we estimated that the concentration of ethanol precipitation product is about 40ng/ul.
Digestion
Digestion to confirm how many PstI cutting sites are in K346007(Ag43 coading) and Ag43-dT complex digestion with SpeI and XbaI.
PstI
DNA solution | 5ul |
PstI | 1ul |
10xH buffer | 2ul |
DW | 12ul |
Total | 20ul |
SpeI and XbaI
DNA solution | 12ul |
SpeI | 1ul |
XbaI | 1ul |
10xH buffer | 2ul |
DW | 4ul |
Total | 20ul |