Team:HokkaidoU Japan/Notebook/aggregation Week 4

From 2012.igem.org

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#Remove supernatant and added 220ul of 70% ethanol.
#Remove supernatant and added 220ul of 70% ethanol.
#Centrifuged in 15000rpm, 10min at 4C.
#Centrifuged in 15000rpm, 10min at 4C.
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#Remove supernatant and air drying in room temperature then added 10ul of DW.  
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#Remove supernatant and air drying in room temperature then added 5ul of DW.  
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Revision as of 08:36, 25 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)

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

DNA solution 5ul
PstI 1ul
10xH buffer 2ul
DW 12ul
Total 20ul


Ag43-dT SpeI and XbaI

DNA solution 12ul
SpeI 1ul
XbaI 1ul
10xH buffer 2ul
DW 4ul
Total 20ul

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.

DNA solution 8ul
HindIII 1ul
10xM buffer 1ul
Total 10ul

July 25th

Digestion

Digestion of pT7-RBS on pSB1C3 cutting with SpeI.

DNA solution 3ul
SpeI 1ul
10xH buffer 1ul
DW 5ul
Total 10ul

[[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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