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

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Team Flavor

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 Reactions
Mira Strep N Mira Strep C Brazz Strep N Brazz Strep C STOP+V0120 BioBrick
diH2O 15 14.5 13.5 11.5 6
Green FD buffer (10x) 2 2 2 2 2
DNA 1 1.5 2.5 4.5 10
EcoR1 1 1 1 1 1
Xba1 - - - - 1
Spe1 1 1 1 1 -


Digestion of Mira/Brazz+Strep and STOP codon BioBrick
Strep+mirabrazz STOP gel.jpg

  • Circled bands were cut and gel purified
   Miraculin + StrepII = 730 bp
   Brazzein + StrepII = 230 bp
   STOP + V0120 = 3200 bp


  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

Ligation Reactions
Mira StrepII N + STOP w/ v0120 BB Mira StrepII C + STOP w/ v0120 BB Brazz StrepII N + STOP w/ v0120 BB Brazz StrepII C + STOP w/ v0120 BB Control STOP w/ v0120 BB
diH2O 4.5 4.5 9 5 12
T4 DNA ligase buffer (10x) 2 3 2 2 2
DNA Insert 7.5 7.5 2 6 0
DNA Backbone 5 5 6 6 5
T4 DNA ligase 1 1 1 1 1


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

File:714failexpcr.jpeg

Arp2 promoter PCR failed.

File:714failarppcr.jpeg

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

Yeast backbone gel purified nanodrops

Sample Concentraion in ng/μL 260/280
31 (1) 8.5 3.00
31(2) 10.2 2.07
32(3) 11.1 2.31
52(4) 28.5 1.83

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

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA 1 uL
AB primer mix (1x) 2.5
Water 35

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