Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

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(RBS digestionのリベンジ)
 
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|style="border-top:1px solid #996"|'''20 uL'''
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
-
→37℃で60分インキュベーション
+
->Incubated at 37C for 60 min
-
* 4 uL 6x Sample Bufferを加え,12 uLずつ電気泳動した
+
* Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed
-
→ゲル抽出<br>
+
->Extracted for gel <br>
-
→10 uLを電気泳動で確認(TSUDA Marker 1)
+
->Electrophoresed 10 uL of Extracted DNA
 +
* Used [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]
-
===だめぽでした!===
+
===Failure===
-
* 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)
+
* 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=
-
===Ligationに必要なDNA Solutionの調製===
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===Preparation of DNA Solution for Ligation===
-
{|style="text-align:center;" class="protocol"
+
{|border="1px" style="text-align:center;" class="protocol"
|-
|-
-
!パーツ
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!Part
-
!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)
!ng/uL
!ng/uL
-
!割合
+
!ratio
-
!size (bp)
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!size(bp)
-
!必要 (ng)
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!required(ng)
-
!使用 (uL)
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!used(uL)
|-
|-
|Vector
|Vector
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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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|}
|}
-
===Ligation & Transformation===
+
===Ligation and [[Team:HokkaidoU_Japan/Protocols|Transformation]]===
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
!Reagent
!Reagent
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|}
|}
-
* 16℃,30 min
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* Incubated at 16C for 30 min
-
* competent cell 50 uLに全量加えた(Transformation)
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* Transformation: Added all to 50 uL of competent cell
-
* 0℃,30 min
+
* Incubated at 0C for 30 min
-
* 42℃,60 sec
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* Heat shocked at 42C for 60 sec
* 5 min on ice
* 5 min on ice
-
* add LB 100 uL
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* Added 100 uL of LB
-
* 37℃,120 min
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* Incubated at 37C for 120 min
-
* spread onto the LBC
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* Spread onto the LBC plate
-
* 37℃,15~20 hrs
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* Incubated at 37C for 20 hrs
-
=RBS 再リベンジ=
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=RBS Retry=
==Digestion==
==Digestion==
{|style="text-align: center" class="protocol"
{|style="text-align: center" class="protocol"
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|style="border-top:1px solid #996"|'''20 uL'''
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
-
→37℃,60 min
+
->Incubated at 37C for 60 min
-
==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
-
* 溶かしてから15,996 rpm @4℃で5分間遠心した
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* Melted and centrifuged at 15000 rpm, 4C for 5 min
-
* 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,15,996 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
-
* 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
-
* 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
-
==電気泳動==
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==Electrophoresis==
-
[[Image:HokkaidoU Japan 20100818b.JPG‎|200px|right|thumb|]]
+
[[Image:HokkaidoU Japan 20100818b.JPG‎|200px|right|thumb| Electrophoresis after Ethanol presipication]]
-
* 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
-
* 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"
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|-
|-
|2
|2
-
|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
|}
|}
-
* 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

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