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Team:HokkaidoU Japan/Notebook/Week 3
From 2012.igem.org
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[[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&P-1.jpg|thumb|Digestion result image]] | [[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&P-1.jpg|thumb|Digestion result image]] | ||
Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp | Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp | ||
| - | We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp. | + | We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp. |
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| + | |||
| + | ===Gel Extraction=== | ||
| + | Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). | ||
| + | Got 50ul of DNA solution. | ||
| + | |||
| + | |||
| + | ===Ethanol Precipitation=== | ||
| + | Ethanol Precipitation for digestion and gel extraction products. | ||
| + | #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 5ul of DW. | ||
| + | |||
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Revision as of 11:09, 16 July 2012
Contents |
July 16th
Digestion
Results of digestion in 15th.
Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.
Gel Extraction
Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.
Ethanol Precipitation
Ethanol Precipitation for digestion and gel extraction products.
- 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 5ul of DW.
