・We took plasmid out of Escherichia coli which have a gene of IL-10 α and IL-10 β and STAT3 in miniprep to use it by a following experiment　(the Escherichia coli　which I really used in an experiment of 2013).
DNA of HlyA and the GFP are IGEM 2014 kit plate1 21G and IGEM 2014 kit plate 13L, so we didn't miniplep

1) We cultured bacterial strain with the LB medium which I added ampicillin to so that density becomes 100ug/ml overnight.(We made a nutrient medium of around 5 ml in 50 ml falcons)(Against 5 ml of nutrient mediums, Amp used 5ul)

2) Aliquot 1ml culture into a 1.5 ml microcentrifuge tube,and Made it spin at 10000rpm (4℃) for 1 min to harvest the bacteria.

3) Removed supernatant and performed 2)operation again, Removed supernatant .

4) Resuspended bacterial pellet by complete vortexing in 100ul SolutionⅠ{D-glucose:9g(50mM),1M Tris-HCl(pH 8.0):25ml(25mM),0.5M EDTA:20ml(10mM),H₂O:955ml /1L}.

5) Inverted bacterial pellet by complete fall mixtureing in 200ul SolutionⅡ{NaOH:8g,SDS:10g[1%(w/v)],H₂O:960ml /1L},and confirmed that it became transparent.

6) Cooled for three minutes in ice.

7) Inverted bacterial pellet by complete fall mixtureing in 150ul SolutionⅢ{CH₃COOH:294.5g(3M),CH₃COOH:120ml(2M),H₂O:diluting in measuring cylinder to 1L total},and confirmed that it became Cloudiness.

8) Cooled for 3 minutes in ice.

9) Harvested the DNA by spinning at 10000rpm (4℃) for 10 min.

10) Gathered only supernatant and moved it in a new microcentrifuge tube.

11) Added 0.8ul Rnase(10mg/ml) and incubate the solution(37℃,1min)

12) Added 200ul phenol:chloroform(1:1) and inverted

13) Harvested by spinning at 10000rpm (4℃) for 5 min.

14) Removed only supernatant and moved it in a new microcentrifuge tube, after that tapped in 200ul chloroform.

15) Harvested by spinning at 10000rpm (4℃) for 1 min.

16) Moved its supernatant to a new microcentrifuge tube and add 15ul 3M CH₃COONa.(Don't gather underlayer)(The ratio of the 3M sodium acetate and supernatant is made to be 10:1)

17) Added 400ul 100%CH₃CH₂OH and made it stirred well.

18) Harvested by spinning at 10000rpm (4℃) for 20 min.

19) Removed only supernatant and added 400ul 70%CH₃CH₂OH(pour a liquid from the other side for white thing not to drain a white.[white thing is plasmid])

20) Harvested by spinning at 10000rpm (4℃) for 20 min.

21) Removed only supernatant and opened the cover of the tube for 10min to dry CH₃CH₂OH.

22) Added 50ul TE to dissolve DNA

23) We stored low temperature

2:PCR

・We performed PCR to confirm whether DNA which we need.

1)We diluted the primer.(D₂W:primer=4:1)

2)Made PCR preparation liquid {buffer×10:2.5ul,dNTP:2ul,PrimerF:1ul,PrimerR:1ul,Taq Polumerase:0.1ul,D₂W:17.39ul /1 microcentrifuge tube(0.2ml)}

3)Added sample(there are Plasmid made with miniprep)to the microcentrifuge tube and do PCR(The PCR conditions are as follows).

Figure①:PCR condition of STAT3 ,IL-10a and IL-10b

[②→③→④]:repeated 35~40 cycles

Figure②:PCR condition of GFP and HlyA

[②→③→④]:repeated 40 cycles

4)We did Electrophoresis to confirm Objective band.

3:Restriction enzyme processing

・We did restriction enzyme processing to plasmid to incorporate inserts in a plasmid vector.

Figure③:Used Restriction enzyme

HlyA・・・We removed the stop codon

F chain ・・・This has EcoR1 recognition sequence.We added CG and A to this to prevent flame out.

・5'-CGGAATTCATTAGCCTATGGAAGTCAGGG-3'

((Tm before adding a restriction enzyme site =60℃

((GC before adding a restriction enzyme site =50.0%

R chain ・・・This has HindⅢ recognition sequence.We added CCC to this.

・5'-CCCAAGCTTTGCTGATGTGGTCAGGGTTA-3'

((Tm before adding a restriction enzyme site =60℃

((GC before adding a restriction enzyme site =50.0%

GFP

F chain ・・・This has HindⅢ recognition sequence.We added CCC and A to this to prevent flame out.

・5'-CCCAAGCTTAATGCGTAAAGGAGAAGAACT-3'

((Tm before adding a restriction enzyme site =56℃

((GC before adding a restriction enzyme site =40.0%

R chain ・・・This has Pst1 recognition sequence.We added AA to this.

・5'-AACTGCAGTTATTATTTGTATAGTTCATCC-3'

((Tm before adding a restriction enzyme site =54℃

((GC before adding a restriction enzyme site =22.7%

Figure④:Rough plan

1)We set a heat block(37℃).

2)prepare one microtube.

3)added 1ul restriction enzyme buffer(×10) to microtube

4)added 1ul DNA sample liquid to the microtube

5)added 1ul each restriction enzyme to the microtube

6)added D₂W until it became 10ul

7)set microtube to a heat block (37℃)

8)left the microtue more than one hour to push forward a reaction

4:Ligation

・We did restriction enzyme processing to plasmid to incorporate inserts in a plasmid vector.

5:Transformation

・We did restriction enzyme processing to plasmid to incorporate GFP+HlyA in a plasmid vector.And we incorporated GFP+HlyA in plasmid by ligation processing

1)We set a water bus(42℃) and a heat block(37℃).

2)added 280ul LB media to empty microtube and set to heat block.

3)added plasmid(1ul or 2ul) to microtube which has 10ul competent cell.

4)Did tap.

5)left this microtube on ice for 15 minutes.

6)did heat shock with a water bus for 45 seconds.

7)brought it to ice quickly and left on ice for 2 min.

8)added 250ul LB media to this.

9)cultured it at 37 ℃ for one hour

10)applied 20ul to LB agar nutrient medium with 20ul and arabinose (200 mg/ml) with ampicillin (100 mg/ml) While I culture it.

11)cultured