IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/21
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Yesterday, we plated transformants for the allergen panel and introns. Today, we will purify the plasmids from the transformants and send any successes in to Genewiz for sequencing.
Concentrations of Allergen Parts and Introns:
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):
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.
Nanodrop for cleaned-up gel purified 5xGalpt negative - ran a 2% E-gel to check. There appears to be something at the .16kb size.
Miraculin 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
OD's V24-1 N/S: 13.5 ng/μL V24-2 N/S: 17.9 ng/μL