IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/12: Difference between revisions

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Revision as of 07:55, 13 July 2010

iGEM iGarden <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Team Allergy

Today, we received sequences for the allergen , intron, and amiRNA inserts. Since 4 of the 8 allergen inserts were not correctly done (and were not set for sequencing) and we want to repeat the inserts that DID work, we will redo ligations for the entire panel.

Procedures

  1. Check Sequencing Results
  2. Redo Ligations for allergen Panel

Results

Sequencing Results

  • amiRNA
    • GFP #1 & GFP.5 #3 worked
    • Bet,LTP did not work
  • intron
    • Pal(#2) matched
  • ihpRNA
    • LTP Sense 1,2,3 and Ger Sense 1 matched
    • Bet sense did not match

Full report (annotated sequences): File:Report.zip

Redo Ligations w/ phosphatized backbone for entire allergen panel,

PME did not grow, we will not use it since we have good PDK and PAL introns.

PAL and PDK will be digested and inserted into a plasmid containing a sense strand of an allergen.

Add PDK to amiRNA vectors if sequences match up


Team Flavor

  • We removed our cultures of e. coli containing Miraculin/Brazzein C/N YFP and NosT & Stop and e. coli containing Miraculin/Brazzein and StrepII tag from the 37°C shaker.
  • Glycerol stocks were made (666 μL glycerol and 333 μL cells)

Miniprep

   ODs
    Miraculin C YFP and NosT & Stop 1: 414.4 ng/μL
    Miraculin C YFP and NosT & Stop 2: 304.0 ng/μL
    Miraculin N YFP and NosT & Stop 1: 428.6 ng/μL
    Miraculin N YFP and NosT & Stop 2: 421.0 ng/μL
    Brazzein C YFP and NosT & Stop 1: 572.8 ng/μL
    Brazzein C YFP and NosT & Stop 2: 580.3 ng/μL
    Brazzein N YFP and NosT & Stop 1: 348.9 ng/μL
    Brazzein N YFP and NosT & Stop 2: 639.6 ng/μL

    Miraculin N StrepII 1: 888.6 ng/μL
    Miraculin N StrepII 2: 614.4 ng/μL
    Miraculin C StrepII 1: 677.1 ng/μL
    Miraculin C StrepII 2: 222.1 ng/μL
    Brazzein N StrepII 1: 352.6 ng/μL
    Brazzein N StrepII 2: 331.6 ng/μL
    Brazzein C StrepII 1: 209.3 ng/μL
    Brazzein C StrepII 2: 346.4 ng/μL


Digestion
Digestion Reactions
Miraculin C YFP NST Miraculin N YFP NST Brazzein N YFP NST Brazzein C YFP NST
diH2O 14 14 13.5 14.3
Green FD buffer (10x) 2 2 2 2
DNA 2 2 2.5 1.7
NotI 1 1 1 1
SpeI 1 1 1 1

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