IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/23

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Team Allergy
Today, we will create our very first biobrick vector with both the sense and intron parts. We will use parts digested yesterday, purify them, and ligate them together.

Procedures

 * 1) Gel Electrophoresis of Intron and Antisense
 * 2) Gel purification of Intron and Antisense inserts
 * 3) Ligation of Intron insert and Sense + V0120
 * 4) Transform and plate

Results
'''Gel Electrophoresis '''

All the Antisense and intron lanes showed, though the last lane (PAL) was faint.

'''Gel Purification '''Nanodrop of gel purification had concentrations of:


 * LTP: 1.8, 2.9
 * Bet v1.1: 6.0, 3.2
 * Bet v1.2: 1.8, 0.5, 1.3
 * Ger 3: 0.8, -0.5
 * Pal: 6.7, 0.7

There is some cause for concern for the lower concentrations, since these small numbers are outside of the range of accuracy.

Ligation of intron insert into Sense + V0120 PME was ligated into all backbones containing sense parts of the allergen panel (8 rxns), followed by PAL ligation into the same backbones. We plated two of each PAL ligation in case that there might be some increased chances of finding growth on plates if more than one plate is done for each ligation. We will check for colonies Saturday.

Genomic DNA extraction from Valencia Oranges

 * We used Qiagen DNeasy Plant Mini Kit
 * 100 mg (wet) sample from Flavedo tissue of orange
 * Samples had little DNA content and dirty 260/280

ODs Orange genomic DNA 1-1: 1.8ng/μL (260/280: .82) Orange genomic DNA 1-2: 1.1ng/μL (260/280: .68) Orange genomic DNA 2-1: 2.6ng/μL (260/280: .85) Orange genomic DNA 2-2: 2.1ng/μL (260/280: .89)


 * PCR ran with between 40ng and 95ng of genomic DNA
 * Extension time increased to 1:30

Mira/Brazz StrepII & Stop

 * Miniprepped per Qiagen protocol and made glycerol stocks


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