Team:HokkaidoU Japan/Notebook/September1

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=1-5A（RFP reporter）のPCR=
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=PCR of 1-5A（RFP reporter）=
{|class="protocol"
{|class="protocol"
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|style="text-align:right;"|'''Reagent'''
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|'''Amount'''
|-
|-
|style="text-align:right;"| Autoclaved DW :  
|style="text-align:right;"| Autoclaved DW :  
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| 1 uL
| 1 uL
|-
|-
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|style="text-align:right;"| Template (1-5A) :  
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|style="text-align:right;"| Template ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]) :  
| 1 uL
| 1 uL
|-
|-
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|style="text-align:right; border-top:1px solid #000;"|'''Total''' :  
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|style="text-align:right; 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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* パーツは1069bp，primerは300bpのため，余裕を持ってExtensionは90秒で行った
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* Because part lenght was 1069 bp plus primer added 300 bp to that, the extension time was 90 sec
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* 得られたPCR solutionをMicrocon YM-10でろ過した
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* PCR product was purified via Microcon YM-10
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===電気泳動===
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[[Image:HokkaidoU Japan 20100901a.JPG‎|200px|right|thumb|]]
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===Electrophoresis===
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pUC119，pSB1C3，1-5Aの濃度を測定するため，電気泳動した（各1 uL使用）
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* 2: λ/''Hin''d III（2 uL)
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[[Image:HokkaidoU Japan 20100901a.JPG‎|200px|right|thumb|Electrophoresis of purified PCR product]]
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* 3: pUC119 (3000bp): 10 ng/uL
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* 4: pSB1C3 (2000bp): 20 ng/uL
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Electrophoresed pUC119，pSB1C3，1-5A to check concentration （1 uL of each）
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* 5: 1-5A (1000bp): 80 ng/uL
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* 2: [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]（2 uL)
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* 3: pUC119 (3000 bp): 10 ng/uL
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* 4: pSB1C3 (2000 bp): 20 ng/uL
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* 5: [[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]] (1000 bp): 80 ng/uL

Latest revision as of 08:01, 27 October 2010

Notebook

PCR of 1-5A（RFP reporter）

Reagent Amount
Autoclaved DW : 33 uL
10x PCR buffer : 5 uL
2 mM dNTPs : 5 uL
25 mM MgSO4 : 3 uL
100 bp up primer : 1 uL
200 bp down primer : 1 uL
KOD plus Neo : 1 uL
Template (1-5A) : 1 uL
Total : 50 uL
  • Because part lenght was 1069 bp plus primer added 300 bp to that, the extension time was 90 sec
  • PCR product was purified via Microcon YM-10

Electrophoresis

Electrophoresis of purified PCR product

Electrophoresed pUC119，pSB1C3，1-5A to check concentration （1 uL of each）

  • 2: λ/Hind III（2 uL)
  • 3: pUC119 (3000 bp): 10 ng/uL
  • 4: pSB1C3 (2000 bp): 20 ng/uL
  • 5: 1-5A (1000 bp): 80 ng/uL
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