Team:HokkaidoU Japan/Notebook/aggregation Week 4

Plasmid extraction

Mini-prep for Ag43(resuspended colony incubated at July 17th and resuspended colony incubated at July 20th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Electrophoresis

Electrophoresis for Ag43(mini-preped at July 23rd) and Ag43 digestion results(digested with EcoRI and SpeI)

Mini-prep result

Digestion result

From 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 at 15000 rpm, 10min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and air drying at room temperature then added 10 ul of DW.

Ethanol precipitation result

From 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 there in K346007 and Ag43-dT complex digested by 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

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

Gel extraction

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

Digestion

Ag43-dT and pSB1AK3 mixture solution(each DNA fragment is about 3kbp) digested with HindIII to digest pSB1AK3.

Digestion

Digestion of pT7-RBS on pSB1K3 digested with SpeI.

Digestion result(Ag43-dT and pT7-RBS)

We were confirmed that pAB1AK3 was digested and became 1.3k and 1.8k bp fragments by HindIII.

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 at 15000 rpm, 10min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 5 min at 4C.
5. Removed supernatant and air drying at room temperature then added 5 ul of DW.

Ligation

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

Transformation

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

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 for 30 min.
4. Apply 1kb DNA ladder and each samples.
5. Ran at 100 V for 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.

Transformation

Transformation of plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) in 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)

Transformation result left:LBC center:LBK right:LBK

There were many colonies on LBC. We guessed 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

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

Digestioned at 37C for 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 at 15000 rpm, 10 min at 4C.
3. Removed supernatant and added 220 ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 10 min at 4C.
5. Removed supernatant and dryed out at room temperature after that added 10 ul of DW.

Ligation

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

Plasmid extraction

Mini-prep for five cultures of 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 confirm the DNA is pT7-RBS-Ag43-dT or not. Plasmid extraction products(pT7-RBS-Ag43-dT)
PstI

Plasmid extraction products(pT7-RBS-Ag43-dT)
EcoR1 and Spe1

Electrophoresis

Electrophoresis for digestion results.

Digestion result

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