Team:Tokyo Metropolitan/Notebook/Pattern/2010/08/25

From 2010.igem.org

(Difference between revisions)
(New page: ==Experiment5：Extraction of a gel== ＜Member＞Mariko, rsk, hitomi ＜Materials＞<br /> ・Thermostable β-Agarase 6 units<br /> ・Agarose gel(after DNA gel electrophoresis)<br /> ...)
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==Experiment5：Extraction of a gel==
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{{:Team:Tokyo_Metropolitan/Header}}
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＜Member＞Mariko, rsk, hitomi
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=2010/08/25=
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＜Materials＞<br />
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・Thermostable β-Agarase 6 units<br />
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・Agarose gel(after DNA gel electrophoresis)<br />
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==1：PCR ==
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＜Member＞<br />
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Mariko, nito
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・Dropped water 400ml<br />
 
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＜Sample＞<br />
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・E-coli (having BBa_I13521 plasmid)
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・E-coli (having BBa_K208017 plasmid)
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・E-coli (having BBa_I732901 plasmid)
＜Protocol＞<br />
＜Protocol＞<br />
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1 Prepared 400ml solution to contain Yeast extract 2.0g, Peptone 2.0g, Glucose 40.0g, NaCl 2.0g and Agerose 6.0g.
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See [https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Pattern/Protocol#PCR_with_Pho_DNA_Polymerase_.28NIPPON_GENE.29/  Protocol 1 ]
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2 Divided up 400ml solution into two Erlenmeyer flasks, and sealed with aluminum. Afterwards , started autoclave(121℃,20min)
 
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3 After finished autoclave, in cleanbench, added 0.2ml Ampcillm to each of the solutions. Afterwards, divided solutions into twenty Petri dishes.
 
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4 Cooled and dried divided solutions in Petri dishes. After Cooling and drying well, kept these in refrigerator.
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・Tube(higher temperature/lower temperature in annealing)
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# Promoter~signal (75.0℃/72.0)
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# cyaA (76.5/75.0)
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# mRFP~Terminator (72.5/71.0)
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# Promoter (76.5/75.0)
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# RBS~signal (76.5/75.0)
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# lacZ (71.0/68.0)
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# Terminator (71.0/68.0)
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# CRP (72.5/71.0)
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Total 16 tubes.
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==2： DNA extraction==
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＜Member＞<br />
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Mariko, nito, Hitomi
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＜Sample＞<br />
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・PCR products
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＜Protocol＞<br />
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See [https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Pattern/Protocol#DNA_extraction/ Protocol 4 ]
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==3：DNA concentration measurement ==
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＜Member＞<br />
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Mariko, nito, Hitomi
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＜Sample＞<br />
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・PCR products
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 +
 
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＜Protocol＞<br />
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# Add 2µl of TE and 2µl of sample into the tube, and mix it gently.
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# Fall in drops 1 on the measuring machine.
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＜Result＞<br />
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{| class="wikitable" style="text-align:center"
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|+ DNA concentration
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!  !! concentration !!  A320  !!  A260/A280  !!  A260/A230 
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|-
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! 1H
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| 165.0 || 0.142 || 1.886 || 0.821
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|-
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! 1L
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| 156.0 || 0.097 || 1.891 || 0.556
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|-
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! 2H
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| 13.5 || 0.059 || 1.868 || 0.341
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|-
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! 2L
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| 15.4 || 0.056 || 2.007 || 0.031
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|-
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! 3H
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| 0.7 || 0.55 || -3.500 || 0.004
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|-
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! 3L
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| -4.8 || 1.02 || 1.484 || -0.025
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|-
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! 4H
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| 10.0 || 0.055 || 1.905 || 0.035
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|-
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! 4L
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| 9.9 || 0.157 || 1.913 || 0.019
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|-
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! 5H
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| -41.0 || 9.39 || 1.464 || 3.475
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|-
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! 5L
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| 0.9 || 0.322 || 0.818 || 0.004
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|-
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! 6H
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| -36.0 || 5.55 || 1.636 || 0.321
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|-
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! 6L
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| 4.5 || 0.86 || 2.282 || 0.050
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|-
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! 7H
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| -29.5 || 6.07 || 1.439 || 2.070
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|-
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! 7L
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| -14.2 || 2.24 || 1.497 || -0.062
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|-
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! 8H
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| 29.5 || 0.169 || 1.934 || 0.051
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|-
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! 8L
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| 26.5 || 0.081 || 1.978 || 0.038
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|}
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==4： Electrophoresis==
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＜Member＞<br />
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Mariko, nito, Hitomi
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＜Sample＞<br />
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・PCR products
 +
 
 +
 
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＜Protocol＞<br />
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See [https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Pattern/Protocol#Electrophoresis/ Protocol 8 ]
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＜Result＞<br />

Revision as of 16:03, 3 September 2010


Contents

2010/08/25

1：PCR

＜Member＞
Mariko, nito


＜Sample＞
・E-coli (having BBa_I13521 plasmid)

・E-coli (having BBa_K208017 plasmid)

・E-coli (having BBa_I732901 plasmid)


＜Protocol＞
See Protocol 1


・Tube(higher temperature/lower temperature in annealing)

  1. Promoter~signal (75.0℃/72.0)
  2. cyaA (76.5/75.0)
  3. mRFP~Terminator (72.5/71.0)
  4. Promoter (76.5/75.0)
  5. RBS~signal (76.5/75.0)
  6. lacZ (71.0/68.0)
  7. Terminator (71.0/68.0)
  8. CRP (72.5/71.0)

Total 16 tubes.


2： DNA extraction

＜Member＞
Mariko, nito, Hitomi


＜Sample＞
・PCR products


＜Protocol＞
See Protocol 4


3：DNA concentration measurement

＜Member＞
Mariko, nito, Hitomi


＜Sample＞
・PCR products


＜Protocol＞

  1. Add 2µl of TE and 2µl of sample into the tube, and mix it gently.
  2. Fall in drops 1 on the measuring machine.


＜Result＞

DNA concentration
concentration A320 A260/A280 A260/A230
1H 165.0 0.142 1.886 0.821
1L 156.0 0.097 1.891 0.556
2H 13.5 0.059 1.868 0.341
2L 15.4 0.056 2.007 0.031
3H 0.7 0.55 -3.500 0.004
3L -4.8 1.02 1.484 -0.025
4H 10.0 0.055 1.905 0.035
4L 9.9 0.157 1.913 0.019
5H -41.0 9.39 1.464 3.475
5L 0.9 0.322 0.818 0.004
6H -36.0 5.55 1.636 0.321
6L 4.5 0.86 2.282 0.050
7H -29.5 6.07 1.439 2.070
7L -14.2 2.24 1.497 -0.062
8H 29.5 0.169 1.934 0.051
8L 26.5 0.081 1.978 0.038



4： Electrophoresis

＜Member＞
Mariko, nito, Hitomi


＜Sample＞
・PCR products


＜Protocol＞
See Protocol 8


＜Result＞

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