Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

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=RBS digestionのリベンジ=
+
=RBS digestion: Revenge=
[[Image:HokkaidoU Japan 20100818a.JPG‎|200px|right|thumb|]]
[[Image:HokkaidoU Japan 20100818a.JPG‎|200px|right|thumb|]]
{|style="text-align: center" class="protocol"
{|style="text-align: center" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|1-2M
|1-2M
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|1 uL
|1 uL
|-
|-
-
|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"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
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→37℃で60分インキュベーション
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->Incubated at 37C for 60 min
-
* 4 uL 6x Sample Bufferを加え，12 uLずつ電気泳動した
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* Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed
-
→ゲル抽出<br>
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->Extracted for gel <br>
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→10 uLを電気泳動で確認（TSUDA Marker 1）
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->Electrophoresed 10 uL of Extracted DNA
 +
* Used [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]
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===だめぽでした！===
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===Failure===
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* おそらくゲル抽出後の吸着でうまくいかず，流れてしまった（小さすぎた）
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* After better inspection of Kit specs it came to lite that retreavel rate for 50 bp and less is 26%
 +
** RBS is just quite small when cut
=Ligation=
=Ligation=
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===Ligationに必要なDNA Solutionの調製===
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===Preparation of DNA Solution for Ligation===
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{|style="text-align:center;" class="protocol"
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{|border="1px" style="text-align:center;" class="protocol"
|-
|-
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|style="border-bottom:1px solid #000;"|パーツ
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!Part
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|style="border-bottom:1px solid #000;"|λ/''Hin''d IIIと比べて (ng/10 uL)
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!By comparison to[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]<br>(ng/10 uL)
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|style="border-bottom:1px solid #000;"|ng/uL
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!ng/uL
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|style="border-bottom:1px solid #000;"|割合
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!ratio
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|style="border-bottom:1px solid #000;"|size (bp)
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!size(bp)
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|style="border-bottom:1px solid #000;"|必要 (ng)
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!required(ng)
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|style="border-bottom:1px solid #000;"|使用 (uL)
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!used(uL)
|-
|-
|Vector
|Vector
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|5 ng/uL
|5 ng/uL
|1
|1
-
|2000 bp
+
|2996 bp
|10 ng
|10 ng
|2 uL
|2 uL
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|0.3 uL
|0.3 uL
|-
|-
-
|double terminator
+
|terminator
|5 ng/10 uL
|5 ng/10 uL
|0.5 ng/uL
|0.5 ng/uL
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|4 uL
|4 uL
|-
|-
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|style="border-top:1px solid #000;" colspan="6"|'''Total'''
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|style="border-top:1px solid #996; text-align:right;" colspan="6"|'''Total'''
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|style="border-top:1px solid #000;"|'''6.3 uL'''
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|style="border-top:1px solid #996;"|'''6.3 uL'''
|}
|}
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===Ligation & Transformation===
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===Ligation and [[Team:HokkaidoU_Japan/Protocols|Transformation]]===
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|DNA solution
|DNA solution
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|T4 ligase
|T4 ligase
|1 uL
|1 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''13.6 uL'''
|}
|}
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* 16℃，30 min
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* Incubated at 16C for 30 min
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* competent cell 50 uLに全量加えた（Transformation）
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* Transformation: Added all to 50 uL of competent cell
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* 0℃，30 min
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* Incubated at 0C for 30 min
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* 42℃，60 sec
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* Heat shocked at 42C for 60 sec
* 5 min on ice
* 5 min on ice
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* add LB 100 uL
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* Added 100 uL of LB
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* 37℃，120 min
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* Incubated at 37C for 120 min
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* spread onto the LBC
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* Spread onto the LBC plate
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* 37℃，15~20 hrs
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* Incubated at 37C for 20 hrs
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+
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=RBS 再リベンジ=
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=RBS Retry=
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Digestion後，エタ沈で回収を試みた
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==Digestion==
{|style="text-align: center" class="protocol"
{|style="text-align: center" class="protocol"
 +
!Reagent
 +
!Amount
|-
|-
|（RBS）1-2M
|（RBS）1-2M
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
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→37℃，60 min
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->Incubated at 37C for 60 min
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==エタ沈==
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==[[Team:HokkaidoU_Japan/Protocols|Ethanol Precipication]]==
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* 2 uLの3 M 酢酸ナトリウムを加えた
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* Added 2 uL of sodium acetate(3 M)
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* 44 uLのエタノールを加えた
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* Added 44 uL of Ethanol
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* 液体窒素の中で凍らせた（'''1.5 mLのチューブに移す'''）
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* Transfered to 1.5 mL tube and frozen with liquid nitrogen
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* 溶かしてから15,000 rpm @4℃で５分間遠心した
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* Melted and centrifuged at 15000 rpm, 4C for 5 min
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* 上清をほかのチューブに移した（一応これも遠心し，上清を捨て，同じ操作をした）
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* Transfered supernatant to another tube
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* 100 uLの70%エタノールで壁をリンスし，15,000 rpm @4℃で５分間遠心した
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** Driven by precaution centrifuged the supernatant and discarded it's supernatant
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* 上清を捨て，真空デシケータで乾燥させた
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* Rinsed the tube walls with 100 uL of 70% Ethanol and centrifuged at 15000 rpm, 4C for 5 min
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* TE 5 uLで溶かした
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* Discarded the supertenant and dried via vacuum desiccator
 +
* Melted in 5 uL of TE
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==電気泳動==
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==Electrophoresis==
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[[Image:HokkaidoU Japan 20100818b.JPG‎|200px|right|thumb|]]
+
[[Image:HokkaidoU Japan 20100818b.JPG‎|200px|right|thumb| Electrophoresis after Ethanol presipication]]
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* TEで溶かしたDNA Solution 1 uLに6x SB 1 uLを加えた
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* Added 1 uL of 6x SB to DNA Solution of 1 uL
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* 最初にとった上清も同じ操作をした
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* Did the same to supernatant　retreaved for precaution
{| class="protocol"
{| class="protocol"
|-
|-
-
|レーン
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|'''Lane'''
-
|
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|'''DNA'''
|-
|-
|2
|2
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|TSUDA Marker 1
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]
|-
|-
|3
|3
-
|上清
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|supernatant
|-
|-
|4
|4
|DNA solution
|DNA solution
|}
|}
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* DNA solutionにしっかり回収されていた
+
* DNA solution band is visible
 +
** Looks OK

Latest revision as of 13:28, 27 October 2010

Notebook

RBS digestion: Revenge

HokkaidoU Japan 20100818a.JPG
Reagent Amount
1-2M 10 uL
DW 4 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

->Incubated at 37C for 60 min

  • Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed

->Extracted for gel
->Electrophoresed 10 uL of Extracted DNA

Failure

  • After better inspection of Kit specs it came to lite that retreavel rate for 50 bp and less is 26%
    • RBS is just quite small when cut

Ligation

Preparation of DNA Solution for Ligation

Part By comparison toλ/Hind III
(ng/10 uL) 		ng/uL ratio size(bp) required(ng) used(uL)
Vector 50 ng/10 uL 5 ng/uL 1 2996 bp 10 ng 2 uL
RFP 250 ng/10 uL 25 ng/uL 2 700 bp 7 ng 0.3 uL
terminator 5 ng/10 uL 0.5 ng/uL 2 200 bp 2 ng 4 uL
Total 6.3 uL

Ligation and Transformation

Reagent Amount
DNA solution 6.3 uL
Ligation solution 6.3 uL
T4 ligase 1 uL
Total 13.6 uL
  • Incubated at 16C for 30 min
  • Transformation: Added all to 50 uL of competent cell
  • Incubated at 0C for 30 min
  • Heat shocked at 42C for 60 sec
  • 5 min on ice
  • Added 100 uL of LB
  • Incubated at 37C for 120 min
  • Spread onto the LBC plate
  • Incubated at 37C for 20 hrs

RBS Retry

Digestion

Reagent Amount
（RBS）1-2M 10 uL
DW 4 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

->Incubated at 37C for 60 min

Ethanol Precipication

  • Added 2 uL of sodium acetate(3 M)
  • Added 44 uL of Ethanol
  • Transfered to 1.5 mL tube and frozen with liquid nitrogen
  • Melted and centrifuged at 15000 rpm, 4C for 5 min
  • Transfered supernatant to another tube
    • Driven by precaution centrifuged the supernatant and discarded it's supernatant
  • Rinsed the tube walls with 100 uL of 70% Ethanol and centrifuged at 15000 rpm, 4C for 5 min
  • Discarded the supertenant and dried via vacuum desiccator
  • Melted in 5 uL of TE

Electrophoresis

Electrophoresis after Ethanol presipication
  • Added 1 uL of 6x SB to DNA Solution of 1 uL
  • Did the same to supernatant　retreaved for precaution
Lane DNA
2 TSUDA Marker 1
3 supernatant
4 DNA solution
  • DNA solution band is visible
    • Looks OK
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August18"