Team:HokkaidoU Japan/Notebook/aggregation Week 6

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Contents

August 6th

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

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

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


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold

mini-prep

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

mini-prep result

transformation

Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3)
in E. coli(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 mini-prep product (pBad-RBS). Because the refine of mini-prep product is not enough.
We use 15 ul DNA solution.

  1. Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 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, 5 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 10 ul of DW.

digestion

digestion of ethanol precipitation product(pBad-RBS).

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

digestion result

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


August 7th

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 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.

Colony PCR

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

DNA solution 4 ul (1colony/10 ul DW)
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer) 0.5 ul
Reverse Primer(PS-R primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53 30
4 72 60
5 72 60
6 4 HOLD

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 in 37C.


August 8th

mini-prep

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

mini-prep result

And then, we did mini-prep for colony No. 2,5,11. The results of them are the same.

Sequencing

Retry for sequencing..

DNA primer
Ag43 mini-prep product 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
K542009 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer
pT7-RBS on pSB1C3 100bp-up forward primer
Ag43-dT on pSB1AK3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
Ag43-dT on pSB1T3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer


Sequencing PCR

template DNA 1 ul
Ready Reaction Premix 1 ul
5x Sequencing Buffer 1.5 ul
H2O 5 ul
Primer(1pmol/ul) 1.5 ul
Total 10 ul


Number Degree Second
1 96 10
2 50 5
3 60 240
4 4 HOLD

Cycle:1~3 x 25


To purify the PCR product, we did ethanol precipitation.


Ethanol precipitation in our sequencing protocol

  1. Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 15 min at 26C.
  3. Remove supernatant and added 100ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 5 min at 26C.
  5. Remove supernatant and air drying in room temperature then added 10 ul of Hi-Di.


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


August 9th

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 DNA (pT7-RBS-Ag43-dT on pSB1K3 ). So, we did ligation by using more Insert DNA part.

pT7-RBS 1 ul
Ag43-dT 4 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold


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