Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

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(→エタ沈)
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* Melted in 5 uL of TE
* 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|]]
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[[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"
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|-
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|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]
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|3
|3
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|上清
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|supernatant
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|4
|4
|DNA solution
|DNA solution
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* DNA solutionにしっかり回収されていた
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* DNA solution band is visible

Revision as of 16:45, 21 September 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 toLambda/Hind III (ng/10 uL) ng/uL ratio size (bp) (ng) required (uL) used
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
double 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
  • ncubated at 37C for 15~20 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(3M)
  • Added 44 uL of Ethanol
  • Transfered to 1.5 mL tube and frozen with liquid nitrogen
  • Melted and centrifuged at 15,996 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 15,996 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
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