Team:KAIT Japan/Protocol

From 2014.igem.org

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<p>
<p>
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:'''HlyA'''<br>
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:<font size="4">'''HlyA'''</font>・・・We removed the stop codon<br>
-
:::F chain  
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::F chain ・・・This has EcoR1 recognition sequence.We added <font color="#FFA500">CG</font> and <font color="#FF0000">A</font> to this to prevent flame out.<br>
 +
:::::・5'-<font color="#FFA500">CG</font>GAATTC<font color="#FF0000">A</font>TTAGCCTATGGAAGTCAGGG-3'<br>
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:::::((Tm before adding a restriction enzyme site =60℃<br>
 +
:::::((GC before adding a restriction enzyme site =50.0%
</p>
</p>
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,p>
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<p>
 +
::R chain ・・・This has HindⅢ recognition sequence.We added <font color="#FFA500">CCC</font> to this.<br>
 +
:::::・5'-<font color="#FFA500">CCC</font>AAGCTTTGCTGATGTGGTCAGGGTTA-3'<br>
 +
:::::((Tm before adding a restriction enzyme site =60℃<br>
 +
:::::((GC before adding a restriction enzyme site =50.0%
 +
</p>
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<p>
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:<font size="4">'''GFP'''</font><br>
 +
::F chain ・・・This has HindⅢ  recognition sequence.We added <font color="#FFA500">CCC</font> and <font color="#FF0000">A</font> to this to prevent flame out.<br>
 +
:::::・5'-<font color="#FFA500">CCC</font>AAGCTT<font color="#FF0000">A</font>ATGCGTAAAGGAGAAGAACT-3'<br>
 +
:::::((Tm before adding a restriction enzyme site =56℃<br>
 +
:::::((GC before adding a restriction enzyme site =40.0%
 +
</p>
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 +
<p>
 +
::R chain ・・・This has Pst1 recognition sequence.We added <font color="#FFA500">AA</font> to this.<br>
 +
:::::・5'-<font color="#FFA500">AA</font>CTGCAGTTATTATTTGTATAGTTCATCC-3'<br>
 +
:::::((Tm before adding a restriction enzyme site =54℃<br>
 +
:::::((GC before adding a restriction enzyme site =22.7%
 +
</p>
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 +
<br>
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<p>
:
:
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Revision as of 06:39, 5 September 2014

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KAIT Japan2013 Kanako.png

KAIT Japan 2014 iGEMRogo.png
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Home

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Team

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Project

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Parts

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Protocol

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Notebook

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Results

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Safety

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Human Practice


Protocol

1:miniprep

・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 I got from miniprep and IGEM kit really increased

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
図1hshshshshshshhshshshshshshshshsh.jpg
[②→③→④]:repeated 35~40 cycles


Figure②:PCR condition of GFP and HlyA
図1がたたたたたt.jpg
[②→③→④]:repeated 40 cycles

4)We did Electrophoresis to confirm Objective band.



3:Restriction enzyme processing

Figure③:Used Restriction enzyme
かか.jpg


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%