Team:HokkaidoU Japan/Notebook/aggregation Week 4

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.

Electrophoresis

Electrophoresis for Ag43(mini-preped above) and Ag43 digestion results(digested with EcoRI and SpeI)

Mini-prep result

Digestion 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.

1. Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
2. Centrifuged in 15000rpm, 10min at 4C.
3. Remove supernatant and added 220ul of 70% ethanol.
4. Centrifuged in 15000rpm, 10min at 4C.
5. Remove supernatant and air drying in room temperature then added 10ul of DW.

Ethanol precipitation result

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. Ag43 PstI

Ag43-dT SpeI and XbaI

Electrophoresis

Electrophoresis for digestion results.

Ag43 d+(P) Digestion result

Ag43-dT d+(X&S) Digestion result

In this result, we found that there are 6 PstI cutting sites in K346007(Ag43).

Gel extraction

Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.

Digestion

Digestion for Ag43-dT and pSB1AK3 mixture(each DNA fragment is about 3kbp) with HindIII to digest pSB1AK3 into about two 1.5kbp fragments.

Digestion

Digestion of pT7-RBS on pSB1C3 cutting with SpeI.

[[image:|thumb|Digestion result(Ag43-dT and pT7-RBS)]]

1. ==Gel extraction==

<p> Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1C3(SpeI). We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.

1. ==Ethanol precipitation==

Ethanol precipitation of digestion and gel extraction products.

1. Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
2. Centrifuged in 15000rpm, 10min at 4C.
3. Remove supernatant and added 220ul of 70% ethanol.
4. Centrifuged in 15000rpm, 10min at 4C.
5. Remove supernatant and air drying in room temperature then added 5ul of DW.

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