Team:HokkaidoU Japan/Notebook/aggregation Week 6

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 dried out at room temperature, after that added 10 ul of DW.

Ligation

We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.

Ligation reaction time was written below.

Plasmid extraction

Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction result

Transformation

Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) in DH5α.

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 hours at 37C.
4. Prepared and Labeled two LBK plates.
5. Plated 300 ul of the culture onto first dish and spread.
6. Added 900 ul of LB to 100 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated them at 37C for 16 hours.

Ethanol precipitation

Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough.
We used 15 ul of all solution.

1. Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 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, 5 min at 4C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

Digestion

Digestion of ethanol precipitation product(pBad-RBS).

digestion result

From this result, speI digested DNA completely.
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.

Gel extraction

Gel extraction for digestion product. 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 dried out at room temperature, after that added 10 ul of DW.

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls. Desired product is about 500~600bp.

Colony PCR result

We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band. Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.

Plasmid extraction

Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction result

And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.

Sequencing

PCR for sequencing.

Sequencing PCR

Cycle:1~3 x 25

To purify the PCR product, we did ethanol precipitation.

Ethanol precipitation for sequencing.

1. Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 15 min at 26C.
3. Removed supernatant and added 100ul of 70% ethanol.
4. Centrifuged at 15000 rpm, 5 min at 26C.
5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)

Ligation

We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th. However, we could not get the target plasmid. (pT7-RBS-Ag43-dT on pSB1K3) So, we did ligation by using more Insert DNA part.

Ligation reaction time was written below.

Transformation

Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5α.
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.

1. Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Added 400 ul of LB then incubated the cells for 2 hours at 37C.
4. Prepared and Labeled two LBK plates.
5. Plated 300 ul of the culture onto first dish and spread.
6. Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated them at 37C for 16 hours.

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 500~600bp.

Colony PCR result

We thought that colonies of No. 6 and 8 is like N2 band. Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.

Plasmid extraction

Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

plasmid extraction result