IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/21

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Team Allergy
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.

Procedures

 * 1) Culture Colonies
 * 2) Miniprep of colonies
 * 3) Diagnostic Digest to check for inserts
 * 4) Send working colonies for sequencing

Results
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):



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



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

 * 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

NEB T4 Ligase was used for all reactions

Digestion & Gel Purification of V24
1039.9 ng of DNA was digested in both reactions
 * The pETDUET Expression Vector, V24, was digested with Not1/Spe1 for later use.

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


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