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 50 ul 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 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 10min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 10 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 10 ul 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 5 ul
PstI 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul


Ag43-dT SpeI and XbaI

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

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 50 ul 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 8 ul
HindIII 1 ul
10xM buffer 1 ul
Total 10 ul


July 25th

Digestion

Digestion of pT7-RBS on pSB1K3 cutting with SpeI.

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

Ethanol precipitation

Ethanol precipitation of digestion and gel extraction products.

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

Ligation

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

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


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 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 200 ul of the transformation onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the transformation and plated 200 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for over 30 hrs.
There were no colony on the plates.

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 5 ul of Etbr and ran at 100 V in 30 min.
  4. Load 1kb DNA ladder and each samples.
  5. Ran at 100 V in 30 min.
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 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled three petri dishes with LBK X2 and LBC.
  5. Plate 300 ul of the transformation onto LBK dish and spread.
  6. Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBC dish and LBK dish then spread.
  7. Incubated the plates at 37C for 17 hrs and 30 min.

Ligation

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

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


Degree Minute
16 30
65 10
4 Hold



July 28th

Transformation result left:LBC center:LBK right:LBK

There were many colonies in LBC! We guess we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.

Liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.

  1. Added 2 ml of LBC into culture tubes.
  2. Resuspended 5 colonies.
  3. Incubated the tubes at 30C for 22 hrs.

Digestion

Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII. pT7-RBS on pSB1K3

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

Ag43-dT on pSB1AK3(cut with SpeI & XbaI)

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

Digestioned at 37C in 2hrs.

digestion result

This results confirmed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we weren't confirmed whether pT7-RBS on pSB1K3 was digested or not.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

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

Ligation

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

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


Degree Minute
16 30
65 10
4 Hold


July 29th

mini-prep

mini-prep for five pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

mini-prep products

Digestion

We need to check the DNA is really pT7-RBS-Ag43-dT or not. mini-prep products(pT7-RBS-Ag43-dT) PstI

DNA solution 4 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


mini-prep products(pT7-RBS-Ag43-dT) EcoR1 and Spe1

DNA solution 4 ul
EcoR1 1 ul
Spe1 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul

Electrophoresis

Electrophoresis for digestion results.

Digestion result

I could not understand what is happend. I tried it again, but the result was the same.