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

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

(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

• 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.

1. Ladder
2. MN2
3. MC2
4. BN2
5. BC2
6. Ladder