Team:HokkaidoU Japan/Notebook/aggregation Week 5

digestion

I tried to digest the mini-prep products again, and after Ethanol precipitation, we did electrophoresis.

digestion result

transformation

I think that the E. coli which we use transformation is BL21(DE3)pLysS. So, the E. coli could grow on LBC plate.
I'm going to do transformation , use DH5α. Transformation of plasmid DNA ligated at 25th (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 200 ul of LB then incubated the cells for 2 hours 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 14 hours.

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

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

mini-prep 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. Single colony isolation on LB plate.
2. Incubated the plate for 15-19 hours at 37C.
3. Lift colony of E. coli into 2 ml LB
4. Incubate at 37C for 12-16 hours at 180-200 rpm
5. Transfer 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.
6. Incubate cells at 20C (for 24 hours over) at 140 rpm
7. Select culture by measuring OD 600.(OD 600=0.5)
8. Incubate the 300 ml flask for 10 min on ice
9. Transfer the culture into two 50 ml Falcon tube
10. Centrifuge at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard sup.
11. Suspend the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)
12. Collect them to one tube.
13. Centrifuge at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard sup.
14. Suspend the pellet in ice-cold 3.2 ml of TB.
15. Instill 0.24 ml of DMSO in precipitant.
16. Incubate the 50 ml Falcon tube for 10 min on ice.
17. Divide 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 in above of thick band. This thick band was same as digestion minus band.

Digestion

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

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 5ul 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 air drying at room temperature then 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.

Transformation

Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in 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. Plate 300 ul of the transformation onto first dish and spread.
6. Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto second dish and spread.
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 No. 5 and 9 colonies were made in exactly transformed in E. coli. and No. 10 colony had high concentration band. Next step, we resuspended these three colonies and cultured.

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

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

mini-prep 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 misdigested band and middle band was digested band 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 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 air drying at room temperature then added 10 ul of Hi-Di.

Then run 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) by 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.