Team:HokkaidoU Japan/Notebook/aggregation Week 4

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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 pSB1K3 cutting with SpeI.

DNA solution 3ul
SpeI 1ul
10xH buffer 1ul
DW 5ul
Total 10ul
Digestion result(Ag43-dT and pT7-RBS)

We were confirmed that pAB1AK3 was digested into 1.3k and 1.8k bp fragments by HindIII. But there are a little doubt SpeI wasn't work because the band of pT7-RBS on pSB1K3 of d- and d+ existed same bp area.

Gel extraction

Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI). We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.

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, 5min at 4C.
  5. Remove supernatant and air drying in room temperature then added 5ul of DW.

Ligation

Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2ul
Ag43-dT 2ul
DW 1ul
Ligation Mighty Mix(TAKARA) 5ul
Total 10ul


Degree Minute
16 30
65 10
4 Hold


July 26th

Transformation

Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).

  1. Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
  2. Incubated on ice for 30min.
  3. Added 200ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 200ul of the transformation onto first dish and spread.
  6. Added 450ul of LB to 50ul of the transformation and plated 200ul of it onto second dish and spread.
  7. Incubated the plates at 37C for OOhrs.

Electrophoresis

Electrophoresis of digestion and ligation products.

  1. Placed TBE agarose gel in Electrophoresis chamber.
  2. Added 1/2X TBE buffer to Electrophoresis chamber.
  3. Added 5ul of Etbr and ran at 100V in 30min.
  4. Load 1kb DNA ladder and each samples.
  5. Ran at 100V in 30min.
Digestion and Ligation results

There are no band in the lane of ligation products. But if digestion products didn't ligate, there are two bands of digestion products would exist in the lane.

July 27th

Transformation

Transformation of plasmid DNA ligated in 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).

  1. Added 2ul of plasmid DNA to 50ul of thawed competent cells on ice.
  2. Incubated on ice for 30min.
  3. Added 600ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK and LBC.
  5. Plate 300ul of the transformation onto LBK dish and spread.
  6. Added 900ul of LB to 100ul of the transformation and plated 300ul of it onto LBC dish and spread.
  7. Incubated the plates at 37C for OOhrs.

Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2ul
Ag43-dT 2ul
DW 1ul
Ligation Mighty Mix(TAKARA) 5ul
Total 10ul


Degree Minute
16 30
65 10
4 Hold