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 AllergyConcentrations of Allergen Parts and Introns:
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 FenceNanodroping 5xGal promoterNanodrop of ligated annealed oligos: 5200.8 ng/μL, 1.32 260/280 E-gel of 5xGal promoterRan 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 promoterpoured 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.
Team FlavorMiraculin N/Brazzein C StrepII & Stop
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
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
NEB T4 Ligase was used for all reactions Digestion & Gel Purification of V24
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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