IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/15

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arabidopsis genomic DNA extraction
Our previous attempt resulted in DNA with the following nanodrop values:


 * 12.1ng/μL 260/280: 0.9
 * 39.1ng/μL 260/280: 1.11

It was believed that the other genomic DNA provided by the Matthews Lab before had similar values with regards to purity/contamination (also approaching 1) Both team allergy and fence performed PCR multiple times unsuccessfully with these DNA samples. It was thought that the low purity may be inhibiting the PCR.

Today we used the QIAgen DNeasy Plant Mini Kit. Liquid nitrogen was poured over the arabidopsis leaves in a mortar while they were ground with the pestle. This produced a fine green powder of arabidopsis leaves from which genomic DNA was extracted starting with step seven of the QIAgen protocol following liquid nitrogen crushing. The only change made to the protocol was that 40 μL of RNase 1 was added per reaction instead of the recommended 4 μL because the concentration of our supply was ten-fold lower.

Nanodrop values were as follows:


 * 6.7 ng/μL 260/280: 1.42
 * 5 ng/μL 260/280: 1.48

Colonies from Ligation of NLS.Serine
Lots of non-specific colonies - ligation failed.





Re-doing Annealing reaction
Placed tube in boiling water for 5 mins, then removed the water from the heat block and allowed the water, and the tube within it, to slowly cool to room temperature.
 * 3μL NLS.Serine.Rev
 * 3μL NLS.Serine.Fwd
 * 2μL 10x Annealing buffer
 * 12μL DH2O

Making Glycerol Stocks

 * making stocks of Barnase 3 (innoculation number 3, miniprep number 1), LacIN1, and Gal4DBD 2
 * Combined .5μL 80% glycerol and .5μL overnight cell culture of the respective bacteria.
 * Vortexed and placed in team fence box of -80°C freezer

10 - #10 on cap, on side: 7-15-10 MPJW B21 backbone Barnase Contains Barnase ligation from pMT1002 PCR 11 - #11 on cap, on side: 7-15-10 MPJW B21 backbone LacINLS Contains: LacI NLS PCR product ligation 12 - #12 on cap, on side: 7-15-10 MPJW B21 Backbone Gal4 Contains: Gal4 DNA Binding Domain

Team Allergy
Today, we did minipreps of intron and backbone. Would have also done minipreps of intron plus allergen part but none of those colonies grew.

We also miniprepped Betv1.2 Sense, Betv1.1 Sense, Bet 1.2 Antisense, and Ger Sense of the plates that we grew on Monday. The rest of the allergen panel did not grow properly. What colonies that WERE on the plate grew probably because AMP was not spread properly on the plate. We deduced this because those colonies could not grow in liquid media. We are waiting on more LB amp plates so that we can redo ligations, which is probably the reason for our lack of colonies.

We will do diagnostic digests of all miniprepped DNA.

Results

 * Minipreps of PDK+V0120 (LTPS+ PDK and Ger_S + PDK did not grow)
 * Made glycerol stocks 500uL of 80% glycerol + 500 uL cells


 * Concentrations of pPDK: 414.2 ng/uL; 478.8 ng/uL; 510.2 ng/uL; 119.2 ng/uL
 * Tomorrow, sending PDK+V0120 for sequencing


 * Looking at sequencing results for our other amiRNA parts
 * Only GFP worked from our first sequencing order. This time, none of the artificial parts had the right sequence
 * Repicking amiRNA colonies to miniprep (6 of each LTP, LTP 0.5, and Bet. We have correct GFP from our first sequencing order)


 * Minipreps of Bet 1S (2 colonies); Bet 1.2 S (1 colony); Bet 1.2 A (1 colony); Ger S (3 colonies)
 * Redoing Ligations & Transformations of Sense/Antisense Parts once we have LB + amp plates
 * Bet 1.1 S: 161 ng/uL & 199.2 ng/uL; Bet 1.2 A: 199.4 ng/uL; Bet 1.2S: 162.5 ng/uL
 * Concentrations of GerS: 347.7 ng/uL; 428.6 ng/uL 541.6 ng/uL


 * Diagnostic Digest of (PDK+V0120; GERS; BetS; Bet 1.2S/A)


 * Ran on gel (Ladder, pdk, V0120 Backbone, Gers (1-3), Bet 1S (1,2), PDK (1-4), Bet 1.2 S, Bet 1.2 A

To send for sequencing: ger s, Bet 1S, PDK 2

Ladder, GFPS, GFPA (These first two should not work), LTPS, LTPA, BetS, BetA, Bet2S, Bet2A, GerS, GerA, PME, PAL, PDK
 * Looking at PCR results for Allergen Panel from Genomic DNA that we extracted (did not work)



Miraculin/Brazzein YFP N/C and NosT + Stop in V24 DUET vector

 * Made glycerol stocks (666 μL glycerol and 333 μL cells)

ODs Miraculin C1: 168.8 ng/μL Miraculin C2: 67.2 ng/μL Miraculin N1: 183.0 ng/μL Miraculin N2: 173.5 ng/μL Brazzein N1: 54.8 ng/μL Brazzein N2: 79.0 ng/μL Brazzein C1: 161.4 ng/μL Brazzein C2: 125.0 ng/μL

Confirmation Digest of Mira/Brazz + YFP + NOSt + STOP in V24 Expression Vector
 * 1) Ladder
 * 2) Mira N1
 * 3) Mira N2
 * 4) Mira C1
 * 5) Mira C2
 * 6) Brazz N1
 * 7) Brazz N2
 * 8) Brazz C1
 * 9) Brazz C2
 * 10) Ladder

Expected Lengths Miraculin + YFP + NOSt + STOP = 700 + 1500 + 250 = 2450 bp    Brazzein + YFP + NOSt + STOP = 300 + 1500 + 250 = 2050 bp     V24 = 5200 bp
 * The N & C designation corresponds to the terminus upon which the YFP was fused.

Team Vector

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