Team:HokkaidoU Japan/Notebook/aggregation Week 5

digestion

I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.

digestion result

Transformation

I think that the E. coli which we used transformation is BL21(DE3)pLysS. So, the E. coli could grow on LBC plate.
I'm going to do transformation , use DH5α. Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into 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 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 spread 200 ul of it onto second dish.
7. Incubated the plates at 37C for 14 hrs.

Liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.

1. Added 2 ml of LBK into culture tubes.
2. Resuspended colonies.
3. Incubated the tubes at 37C for 18 hrs.

Plasmid extraction

Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction result

Digestion

pT7-RBS(20 ng/ul) =No. 9
SpeI using 10xH

SpeI using 10xM

pT7-RBS(30 ng/ul) =No. 10
SpeI using 10xH

SpeI using 10xM

digestion result

We couldn't digested them exactly, so we tried to digest once more time.

pT7-RBS(20 ng/ul) =No. 9
SpeI 10xH

Liquid culture

Liquid culture for pBAD-RBS on pSB1K3.

1. Added 2 ml of LBK into culture tube.
2. Scraped the surface of glycerol stock of construct.
3. Incubated the tube at 33C.

Preparing chemical competent cell

Preparing chemical competent cell of BL21, JM109 and DH5α. Chemical competent cell made in each E. coli strains.

Our competent cell Protocol

1. Did single colony isolation on LB plate.
2. Incubated the plate for 15-19 hours at 37C.
3. Lifted colony of E. coli into 2 ml LB.
4. Incubated at 37C for 12-16 hrs at 180-200 rpm.
5. Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.
6. Incubated cells at 20C (over 24 hrs) at 140 rpm.
7. Selected culture by measuring OD 600.
8. Incubated the 300 ml flask for 10 min on ice.
9. Transfered the culture into two 50 ml Falcon tubes.
10. Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
11. Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)
12. Collected them to one tube.
13. Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.
14. Suspended the pellet in ice-cold 3.2 ml of TB.
15. Instilled 0.24 ml of DMSO in precipitant.
16. Incubated the 50 ml Falcon tube for 10 min on ice.
17. Divided 50 ul of solutions in each 0.5 ml tubes.
18. Freezed the suspension by liquid nitrogen.
19. Stored at –80C.

Electrophoresis

Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)

Electrophoresis result

There were low concentration band above thick band. This thick band was same as digestion minus band.

Digestion

Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.

Electrophoresis

Electrophoresis for the result of digestion.

Electrophoresis result

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 125ul of 100% ethanol.
2. Centrifuged at 15000 rpm, 10 min at 4C.
3. Removed supernatant and added 220ul 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.

Ligation

Ligation of 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.

Transformation

Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5α.

1. Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30min.
3. Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
4. Prepared and Labeled two petri dishes with LBK.
5. Spread 300 ul of the transformation onto first dish.
6. Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish.
7. Incubated the plates at 37C for 15 hrs 45 min.

PCR

PCR to confirm Ag43-f4 primer.

Cycle:2~4 x 45

PCR result

From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.

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 No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band. Next step, we resuspended these three colonies and incubated.

Liquid culture

Liquid culture of colonies passed the colony PCR test.

1. Added 2 ml of LBK into culture tubes.
2. Resuspended 3 colonies.
3. Incubated the tubes at 37C for 13 hrs.

Plasmid extraction

Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction results

Digestion

Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI. pT7-RBS on pSB1K3

pT7-RBS (once digestioned with SpeI)

digestion result

From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?

Gel extraction

Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Sequencing

Sequencing to confirm what kind of DNA we made.

Sequencing PCR

Cycle:1~3 x 25

To extract 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 Hi-Di.

Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)
We could not get the sequencing data.

Digestion

Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.

digestion result

From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.

Gel extraction

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