Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August17|August 17]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August19|August 19]]</div></div>
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=RBS digestion: Revenge=
 +
[[Image:HokkaidoU Japan 20100818a.JPG‎|200px|right|thumb|]]
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{|style="text-align: center" class="protocol"
 +
!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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''20 uL'''
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|}
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->Incubated at 37C for 60 min
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* Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed
 +
->Extracted for gel <br>
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->Electrophoresed 10 uL of Extracted DNA
 +
* Used [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]
 +
 
 +
===Failure===
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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
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 +
=Ligation=
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===Preparation of DNA Solution for Ligation===
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{|border="1px" style="text-align:center;" class="protocol"
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|-
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!Part
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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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!ng/uL
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!ratio
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!size(bp)
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!required(ng)
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!used(uL)
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|-
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|Vector
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|50 ng/10 uL
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|5 ng/uL
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|1
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|2996 bp
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|10 ng
 +
|2 uL
 +
|-
 +
|RFP
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|250 ng/10 uL
 +
|25 ng/uL
 +
|2
 +
|700 bp
 +
|7 ng
 +
|0.3 uL
 +
|-
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|terminator
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|5 ng/10 uL
 +
|0.5 ng/uL
 +
|2
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|200 bp
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|2 ng
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|4 uL
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|-
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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 #996;"|'''6.3 uL'''
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|}
 +
 
 +
===Ligation and [[Team:HokkaidoU_Japan/Protocols|Transformation]]===
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{|style="text-align:center;" class="protocol"
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!Reagent
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!Amount
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|-
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|DNA solution
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|6.3 uL
 +
|-
 +
|Ligation solution
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|6.3 uL
 +
|-
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|T4 ligase
 +
|1 uL
 +
|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''13.6 uL'''
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|}
 +
 
 +
* Incubated at 16C for 30 min
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* Transformation: Added all to 50 uL of competent cell
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* Incubated at 0C for 30 min
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* Heat shocked at 42C for 60 sec
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* 5 min on ice
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* Added 100 uL of LB
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* Incubated at 37C for 120 min
 +
* Spread onto the LBC plate
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* Incubated at 37C for 20 hrs
 +
 
 +
=RBS Retry=
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==Digestion==
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{|style="text-align: center" class="protocol"
 +
!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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
 +
->Incubated at 37C for 60 min
 +
 
 +
==[[Team:HokkaidoU_Japan/Protocols|Ethanol Precipication]]==
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* Added 2 uL of sodium acetate(3 M)
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* 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==
 +
[[Image:HokkaidoU Japan 20100818b.JPG‎|200px|right|thumb| Electrophoresis after Ethanol presipication]]
 +
* Added 1 uL of 6x SB to DNA Solution of 1 uL
 +
* Did the same to supernatant　retreaved for precaution
 +
 
 +
{| class="protocol"
 +
|-
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|'''Lane'''
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|'''DNA'''
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|-
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|2
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]
 +
|-
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|3
 +
|supernatant
 +
|-
 +
|4
 +
|DNA solution
 +
|}
 +
* 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"