IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/15: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 55: Line 55:


*Ran on gel (Ladder, pdk, V0120 Backbone, Gers (1-3), Bet 1S (1,2), PDK (1-4), Bet 1.2 S, Bet 1.2 A
*Ran on gel (Ladder, pdk, V0120 Backbone, Gers (1-3), Bet 1S (1,2), PDK (1-4), Bet 1.2 S, Bet 1.2 A
[[Image:|240 px]]
[[Image:2010-07-15_16hr_33min.jpg|240 px]]
 
To send for sequencing: ger s, Bet 1S, PDK 2


*Looking at PCR results for Allergen Panel from Genomic DNA that we extracted (did not work)
*Looking at PCR results for Allergen Panel from Genomic DNA that we extracted (did not work)

Revision as of 13:36, 15 July 2010

iGEM iGarden <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Team Allergy

Today, we did minipreps of intron and backbone. Would have also done minipreps of intron plus allergen part but none of those colonies grew.

We also miniprepped Betv1.2 Sense, Betv1.1 Sense, Bet 1.2 Antisense, and Ger Sense of the plates that we grew on Monday. The rest of the allergen panel did not grow properly. What colonies that WERE on the plate grew probably because AMP was not spread properly on the plate. We deduced this because those colonies could not grow in liquid media. We are waiting on more LB amp plates so that we can redo ligations, which is probably the reason for our lack of colonies.

We will do diagnostic digests of all miniprepped DNA.


Results

  • Minipreps of PDK+V0120 (LTPS+ PDK and Ger_S + PDK did not grow)
    • Made glycerol stocks 500uL of 80% glycerol + 500 uL cells
    • Concentrations of pPDK: 414.2 ng/uL; 478.8 ng/uL; 510.2 ng/uL; 119.2 ng/uL
      • Tomorrow, sending PDK+V0120 for sequencing
  • Looking at sequencing results for our other amiRNA parts
    • Only GFP worked from our first sequencing order. This time, none of the artificial parts had the right sequence
    • Repicking amiRNA colonies to miniprep (6 of each LTP, LTP 0.5, and Bet. We have correct GFP from our first sequencing order)
  • Minipreps of Bet 1S (2 colonies); Bet 1.2 S (1 colony); Bet 1.2 A (1 colony); Ger S (3 colonies)
    • Redoing Ligations & Transformations of Sense/Antisense Parts once we have LB + amp plates
    • Bet 1.1 S: 161 ng/uL & 199.2 ng/uL; Bet 1.2 A: 199.4 ng/uL; Bet 1.2S: 162.5 ng/uL
    • Concentrations of GerS: 347.7 ng/uL; 428.6 ng/uL 541.6 ng/uL
  • Diagnostic Digest of (PDK+V0120; GERS; BetS; Bet 1.2S/A)
Digestions
pPDK (1-4) pGERSPDK (1-3) Bet1S (2); Bet2S/A
DNA 1 1 1
FD Buffer (10x) 2 2 2
diH2O 15 15 15
Xba1 1 1 1
Pst 1 1 1
  • Ran on gel (Ladder, pdk, V0120 Backbone, Gers (1-3), Bet 1S (1,2), PDK (1-4), Bet 1.2 S, Bet 1.2 A

To send for sequencing: ger s, Bet 1S, PDK 2

  • Looking at PCR results for Allergen Panel from Genomic DNA that we extracted (did not work)

Ladder, GFPS, GFPA (These first two should not work), LTPS, LTPA, BetS, BetA, Bet2S, Bet2A, GerS, GerA, PME, PAL, PDK

Team Fence

Team Flavor

Miraculin/Brazzein YFP N/C and NosT + Stop in V24 DUET vector

  • Made glycerol stocks (666 μL glycerol and 333 μL cells)
   ODs
    Miraculin C1: 168.8 ng/μL
    Miraculin C2: 67.2 ng/μL
    Miraculin N1: 183.0 ng/μL
    Miraculin N2: 173.5 ng/μL
    Brazzein N1: 54.8 ng/μL
    Brazzein N2: 79.0 ng/μL
    Brazzein C1: 161.4 ng/μL
    Brazzein C2: 125.0 ng/μL
Confirmation Digestion Reactions
Mira N1 Mira N2 Mira C1 Mira C2 Brazz N1 Brazz N2 Brazz C1 Brazz C2
diH2O 13.5 13.5 13.5 11 11 11 13.5 13
Green FD buffer (10x) 2 2 2 2 2 2 2 2
DNA 2.5 2.5 2.5 5 5 5 2.5 3
PstI 1 1 1 1 1 1 1 1
Xba1 1 1 1 1 1 1 1 1


Confirmation Digest of Mira/Brazz + YFP + NOSt + STOP in V24 Expression Vector

  1. Ladder
  2. Mira N1
  3. Mira N2
  4. Mira C1
  5. Mira C2
  6. Brazz N1
  7. Brazz N2
  8. Brazz C1
  9. Brazz C2
  10. Ladder
  • The N & C designation corresponds to the terminus upon which the YFP was fused.
   Expected Lengths
    Miraculin + YFP + NOSt + STOP = 700 + 1500 + 250 = 2450 bp
    Brazzein + YFP + NOSt + STOP = 300 + 1500 + 250 = 2050 bp
    V24 = 5200 bp

Team Vector


Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Harvard/2009/Notebook/Harvard_iGEM_2010/2010/07/15&oldid=433280"