IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/21: Difference between revisions

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* We ran a confirmation digest on the miniprepped Miraculin N StrepII & Stop and the Brazzein C StrepII & Stop. We digested the samples with XbaI/PstI.  We should have used NotI/SpeI in this digest if we planned on inserting into V24 pETDUET vector.   
* We ran a confirmation digest on the miniprepped Miraculin N StrepII & Stop and the Brazzein C StrepII & Stop. We digested the samples with XbaI/PstI.  We should have used NotI/SpeI in this digest if we planned on inserting into V24 pETDUET vector.   
**We digested 558 ng of Mira N1, 516 ng Mira N2, 540 ng Brazz C1, and 551 ng Brazz C2 in 20 μL digestion reactions.  
**We digested 558 ng of Mira N1, 516 ng Mira N2, 540 ng Brazz C1, and 551 ng Brazz C2 in 20 μL digestion reactions.  


* We also ran undigested samples of all four to help determine why the other ligations (Brazz N and Mira C) did not work.  
* We also ran undigested samples of all four to help determine why the other ligations (Brazz N and Mira C) did not work.  
**We ran 279 ng of Mira N1, 258 ng Mira N2, 270 ng of Brazz C1, and 275.5 ng Brazz C2.
**We ran 279 ng of Mira N1, 258 ng Mira N2, 270 ng of Brazz C1, and 275.5 ng Brazz C2.


Revision as of 14:09, 21 July 2010

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Team Allergy

Concentrations of Allergen Parts and Introns:

  • Allergen Parts:
    • Sense:
    • Antisense:
  • Intron (Pal):

Diagnostic Digest

lader; ladder; GerS (1-3); Ger A (1-3); Bet2S (1-3); Bet2A (1-3); BetA (!-3); LTPS (1-3); LTPA (1-3); Pal (1-3); ladder; ladder

(More contrasted image below):


Team Fence

Nanodroping 5xGal promoter

Nanodrop of ligated annealed oligos: 5200.8 ng/μL, 1.32 260/280

E-gel of 5xGal promoter

Ran 2% E-gel of .5μL of the ligation reaction (2.6μg) in 15μL water, with 10μL of KB plus ladder on either side.

Ladder ran screwy, so new ladder used,and a 2% agarose Gel was poured for gel extraction

Gel Extraction of 5xGal promoter

poured 2% agarose Gel for gel extraction, ran 2μL of the ligated annealing reaction in 13μL EB buffer, with two KB plus 1 ladders, one on each side.


Gel extraction performed, into an eppendorf weighing .9563g

Team Flavor

Miraculin N/Brazzein C StrepII & Stop

  • We ran a confirmation digest on the miniprepped Miraculin N StrepII & Stop and the Brazzein C StrepII & Stop. We digested the samples with XbaI/PstI. We should have used NotI/SpeI in this digest if we planned on inserting into V24 pETDUET vector.
    • We digested 558 ng of Mira N1, 516 ng Mira N2, 540 ng Brazz C1, and 551 ng Brazz C2 in 20 μL digestion reactions.
  • We also ran undigested samples of all four to help determine why the other ligations (Brazz N and Mira C) did not work.
    • We ran 279 ng of Mira N1, 258 ng Mira N2, 270 ng of Brazz C1, and 275.5 ng Brazz C2.


Gel 


   Gel Lanes:
    1. 1kb plus ladder
    2. Mira N1 XbaI/PstI
    3. Mira N2 XbaI/PstI
    4. Brazz C1 XbaI/PstI
    5. Brazz C2 XbaI/PstI
    6. 1kb plus ladder
    7. Mira N1 undigested
    8. Mira N2 undigested
    9. Brazz C1 undigested
   10. Brazz C2 undigested
  • Gel shows bands consistent with successful ligation!
  • Gel extraction and purification of Miraculin, StrepII & Stop insert and Brazzein, StrepII & Stop insert.
   ODs
    Mira N1: 5.4 ng/μL
    Mira N2: 3.1 ng/μL
    Brazz C1: 6.1 ng/μL
    Brazz C2: 10.1 ng/μL


Ligation/Transformation of Mira/Brazz+StrepII

  • Our Miraculin and Brazzein constructs with StrepII tags (pre-cut with EcoR1/Spe1) were re-ligated to STOP+V0120 backbone
Ligation Reactions
Insert DNA BackBone DNA
Mira N 9 ng 49.5 ng
Mira C 22.4 ng 49.5 ng
Brazz N 37.4 ng 49.5 ng
Brazz C 14.3 ng 53.4 ng
Control 0 ng 53.4 ng

NEB T4 Ligase was used for all reactions

Digestion & Gel Purification of V24

  • The pETDUET Expression Vector, V24, was digested with Not1/Spe1 for later use.

1039.9 ng of DNA was digested in both reactions

Digestion reactions were run on a 1% Agarose gel at 125V for 30 min 

   Specs
    V24-1 N/S: 13.5 ng/μL
    V24-1 N/S: 17.9 ng/μL



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