Team:HokkaidoU Japan/Notebook/September3

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=前日のトラフォメの結果=
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=Results of yesterdays transformation=
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* pSB1C3を使ったトラフォメは全滅だった
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* pSB1C3 uterly failed to produce colonies
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* pUC119では計20個ほどのコロニーしか得られなかった
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* pUC119 produced 20 colonies
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* 赤くなるはずが，赤くなっていなかったので，目的のInsertが入っていない可能性がある．
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* colonies that should been red because of RFP insert wasn't, so there is possibility that insert wasn't there
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=前日のトラフォメ菌の[[コロニーPCR]]=
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=Colony PCR on yesterdays E.coli=
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* プロトコル：コロニーPCRに従って，実験した
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* Colony PCR was done according to protocol
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* 今回は20サンプルで行った
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* This day we did 20 samples
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===電気泳動===
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===Electrophoresis===
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=Concentration check of parts used for transformation yesterday=
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[[Image:HokkaidoU_Japan_20100903a.jpg|200px|right|thumb|Electrophoresis of parts before ligation]]
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Confirmed that DNA solution concentration of parts for Ligation was as anticipated (good?)
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=前日に使用したパーツの濃度測定=
 
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[[Image:HokkaidoU_Japan_20100903a.jpg|200px|right|thumb|]]
 
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Ligationに使用したDNA solutionが予想通りの濃度だったのか確認した
 
{|class="protocol"
{|class="protocol"
|-
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|-
|-
|2
|2
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|λ/Hind III, EcoR I
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/Hind III, EcoR I]
|-
|-
|3
|3
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|}
|}
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=1-3AのPCR=
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=[[Team:HokkaidoU_Japan/Protocols|PCR]] of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]]=
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1-3AはpSB1C3に載ったRFP reporterで，トラフォメに成功している<br>
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ベクターとして配布されたpSB1C3が悪い可能性を見るため，このパーツのpSB1C3を増幅して使用する
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[[Team:HokkaidoU_Japan/Protocols|Transformation]] of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] part which is originaly on pSB1C3 was succesful<br>
 +
Thinking that linearized vector might gone bad we PCRed pSB1C3 from [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]]
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{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
!Reagent
!Reagent
!Amount
!Amount
|-
|-
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|1-3A
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]]
|1
|1
|-
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|1
|1
|-
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''50 uL'''
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|style="border-top:1px solid #996;"|'''50 uL'''
|}
|}
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* extensionは120 sec
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* Extension was for120 sec
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* YM-10でClean Upしたら43 uLになった
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* Purified via Microcon YM-10
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* Final volume was 43 uL

Latest revision as of 08:07, 27 October 2010

Notebook

Results of yesterdays transformation

  • pSB1C3 uterly failed to produce colonies
  • pUC119 produced 20 colonies
  • colonies that should been red because of RFP insert wasn't, so there is possibility that insert wasn't there

Colony PCR on yesterdays E.coli

  • Colony PCR was done according to protocol
  • This day we did 20 samples

Electrophoresis

Concentration check of parts used for transformation yesterday

Electrophoresis of parts before ligation

Confirmed that DNA solution concentration of parts for Ligation was as anticipated (good?)

Lane DNA
1
2 λ/Hind III, EcoR I
3
4 RFP
5 pUC119
6 pSB1C3

PCR of 1-3A

Transformation of 1-3A part which is originaly on pSB1C3 was succesful
Thinking that linearized vector might gone bad we PCRed pSB1C3 from 1-3A

Reagent Amount
1-3A 1
DW 33
10x Buffer 5
2 M 4dNTPs 5
25 mM MgSO4 3
Suffix-F 1
Prefix-R 1
KOD 1
Total 50 uL
  • Extension was for120 sec
  • Purified via Microcon YM-10
  • Final volume was 43 uL
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