IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/27

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(Team Flavor)
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==Team Flavor==
==Team Flavor==
 +
===Ligation of Mira/Brazz + StrepII to STOP + V0120===
 +
*Insert to Backbone ration of 3:1
 +
    Miraculin: 34ng
 +
    Brazzein:  11ng
 +
    Backbone:  50ng
 +
 +
*Ligations were transformed, plated onto LB+AMP and left overnight @ 37°C
 +
 +
===PCR of Valencene using Pfx Polymerase===
 +
*Pfx polymerase was rumoured to be more accurate at PCR from genomic DNA
 +
    Specs:
 +
    DNA 1-1: 1.8 ng/μL
 +
    DNA 1-2: 1.1 ng/μL
 +
    DNA 2-1: 2.6 ng/μL
 +
    DNA 2-2: 2.2 ng/μL
 +
 +
*1μL of each DNA sample was used for PCR.  Cycling procedure can be found here: http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.79411.File.dat/platinumpfx_pps.pdf
 +
===Mira/Brazz+StrepII+STOP confirmation digest===
 +
    Digested DNA:
 +
    MN2: 211.2 ng
 +
    MC2: 190.2 ng
 +
    BN2:  184.4 ng
 +
    BC2:  219.2 ng
 +
 +
*Digestions were run on a 1% agarose gel at 125V for 30 minutes.
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Revision as of 17:20, 27 July 2010

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Team Allergy

  • Diagnostic Digest (EcoR1 & Pst1)

1-6: LTP + PDK (3200 & 1200);

7-16: LTP + PAL (3200 & 540);

17,18: Bet1 + PDK (3200 & 1200);

19-22: Bet1 + PAL (3200 & 540);

23,24: Bet2+PDK (3200 & 1200);

25-28: Bet2 + PAL (3200 & 540);

29-32: Ger3 + PDK (3200 & 1200);

33-38: Ger3 + PAL (3200 & 540);

Image: Ladder (every 6)

Rerun:

  • Two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)
    • Digested these parts with spe and pst1 (slow digest)

Antisense-Intron Backbone Digestion

1-39
DNA ~1.5 ug
10x BSA 3
diH2O Fill to 30
Spe 1.5
Pst1 1.5
Neb Buffer 2 (10x) 3

(Let it go for 2 hours and then phosphatized backbone (20 min)

  • Ligated with antisense parts and transformed

Team Flavor

Ligation of Mira/Brazz + StrepII to STOP + V0120

  • Insert to Backbone ration of 3:1
   Miraculin: 34ng
   Brazzein:  11ng
   Backbone:  50ng
  • Ligations were transformed, plated onto LB+AMP and left overnight @ 37°C

PCR of Valencene using Pfx Polymerase

  • Pfx polymerase was rumoured to be more accurate at PCR from genomic DNA
   Specs:
    DNA 1-1: 1.8 ng/μL
    DNA 1-2: 1.1 ng/μL
    DNA 2-1: 2.6 ng/μL
    DNA 2-2: 2.2 ng/μL

Mira/Brazz+StrepII+STOP confirmation digest

   Digested DNA:
    MN2: 211.2 ng
    MC2: 190.2 ng
    BN2:  184.4 ng
    BC2:  219.2 ng
  • Digestions were run on a 1% agarose gel at 125V for 30 minutes.


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