Team:HokkaidoU Japan/Notebook/August18

From 2010.igem.org

(Difference between revisions)
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|style="border-top:1px solid #996"|'''20 uL'''
|style="border-top:1px solid #996"|'''20 uL'''
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→Incubated at 37C for 60 min
+
->Incubated at 37C for 60 min
* Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed  
* Added 4 uL of 6x Sample Buffer making it total of 12 uL per lane and electrophoresed  
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→Extracted for gel <br>
+
->Extracted for gel <br>
-
→Electrophoresed 10 uL of Extracted DNA
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->Electrophoresed 10 uL of Extracted DNA
* Used [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]  
* Used [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png TSUDA Marker 1]  
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|style="border-top:1px solid #996"|'''20 uL'''
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
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→Incubated at 37C for 60 min
+
->Incubated at 37C for 60 min
==[[Team:HokkaidoU_Japan/Protocols|Ethanol Precipication]]==
==[[Team:HokkaidoU_Japan/Protocols|Ethanol Precipication]]==

Revision as of 13:25, 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 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 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