IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/14

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Miniprep of STOP + V0120 part
Nanodrop O/D's:   STOP V0120 1-1: 134.4 ng/μL STOP V0120 1-2: 98.7 ng/μL STOP V0120 2-1: 55.2 ng/μL STOP V0120 2-2: 121.8 ng/μL

DTL of StrepII+Mira/Brazz with STOP codon

 * To insert a STOP codon to the end of our Mira/Brazz StrepII constructs, we used the STOP+V0120 BioBrick as our vector (EcoR1/Xba1 digest) and our Miraculin/Brazzein construct as our insert (EcoR1/Spe1 digest)
 * Construct DNA used was from Miniprep 1 (Box 4)
 * ~1μg of DNA was digested in each reaction

Digestion of Mira/Brazz+Strep and STOP codon BioBrick Miraculin + StrepII = 730 bp   Brazzein + StrepII = 230 bp    STOP + V0120 = 3200 bp
 * Circled bands were cut and gel purified


 * 1) 1kb Plus Ladder
 * 2) Miraculin C Strep E/S
 * 3) Miraculin N Strep E/S
 * 4) Brazzein C Strep E/S
 * 5) Brazzein N Strep E/S
 * 6) STOP + V0120 E/X
 * 7) 1kb Plus Ladder

Gel Purification Specs Mira N StrepII E/S: 3.0 ng/μL Mira C StrepII E/S: 2.8 ng/μL Brazz N StrepII E/S: 3.4 ng/μL Brazz C StrepII E/S: 1.3 ng/μL STOP+V0120 BB E/X: 7.1 ng/μL

Ligation of Mira/Brazz+Strep and STOP codon BioBrick

Transformation


 * Transformed 5 μL ligation mix with 15 μL TURBO e. coli cells
 * Plated on LB + Amp plates and left in 37°C incubator overnight

Team Fence
PCR of the arabidopsis promoters with the newly extracted DNA and the older DNA. FAIL



Arp2 promoter PCR failed.



Exp2 promoter PCR failed.

Phenol Chloroform DNA Extraction

 * resuspend DNA in approximately 200μL molecular grade pure water
 * add 1 volume phenol chloroform
 * when pipetting the phenol chloroform be sure to pipette from closer to the middle level of the liquid as the top two centimeters do not contain what we want
 * centrifuge at top speed for 5 minutes
 * collect the aqueous top layer and transfer to a clean microcentrifuge tube
 * the remaining liquid is toxic and must be carefully disposed of in the "toxic waste" bottle in the fume hood
 * add 1 volume phenol chloroform
 * centrifuge at top speed for 5 minutes
 * collect the aqueous top layer and put into a fresh tube
 * add three volumes 100% ethanol and 1/10 volume sodium acetate
 * allow solution to incubate on ice for 15 minutes
 * centrifuge at top speed for 10 minutes
 * pipette off the aqueous solution leaving a pellet of DNA in the bottom of the tube
 * wash in 500μL 70% ethanol
 * mix tube by inverting
 * centrifuge again for 10 minutes at top speed
 * air dry pellet by leaving tube open on bench (not on ice) til dry
 * after pellet has dried resuspend DNA in desired volume of H2O or buffer EB
 * presence of DNA can be confirmed by running a small sample on a gel

THE END

NLS Ligations
Ligation of NLS.Serine
 * 2.5μL Backbone
 * 1.5μL of 1/100 dilution (1.5ng NLS.Serine)
 * 6μL H2O
 * 10μL 2x quick ligase buffer
 * 1μL quick ligase

B21 control
 * 2.5μL B21 Backbone
 * 7.5μL DH2O
 * 10μL 2x Quick Ligase Buffer
 * 1μL Quick Ligase

Transformation

 * 1μL NLS lig into TOP10 cells
 * Chilled on ice for 30 mins
 * Heat shocked at 42°C for 30 seconds
 * Put on ice for 2 mins
 * Added 170μL SOC medium
 * Set to shake in incubator for 30 mins
 * Streaked on LB+amp plates, incubated overnight

Team Allergy

 * Genomic DNA extraction
 * PCR for allergen panel from genomic DNA
 * Grew up cultures of transformations from yesterday (LTPS+intron; GerS+intron; PDK+V0120)--tubes weren't cloudy so we grew overnight cultures again

8 reactions (Ger S/A; BetS/A; Bet1.2 S/A; LTP S/A)

Annealing Temp: 65 C Extension Temp: 30 sec

Concentrations: BetS: 109.1 ng/uL; BetA: 90.5 ng/uL; GerS: 69 ng/uL; GerA: 46 ng/uL; LTPS:36.7 ng/uL; LTPA:5.2 ng/uL; Bet1.2S: 77.4 ng/uL; Bet1.2A:66.2 ng/uL; PME: 64.2 ng/uL; PAL: 62.9 ng/uL; PDK: 35.7 ng/uL; PDK(2): 81.3 ng/uL; PDK(3): 21.5 ng/uL


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