IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/06/30

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June 30, 2009

  • Inoculated YPD media with overnight culture, 1:50 dilution
  • Overnight culture OD=1.754 (wavelength 660nm)
10mL of YPD media+.2μL overnight culture
  • DNA wasn't ready so didn't make competent cells today. Can leave O/N culture out until we're ready to use it (hopefully tomorrow)

PCR

  • Ran 1.2% agarose gel of overnight PCR products
Gel 1: 1 kb Ladder, -DNA, -Enz, 035, 139, 049, 186
Gel 2: 1 kb Ladder, -DNA, -Enz, 213, 236, 346

2009-06-30.Longtine0.35-186.jpg 2009-06-30.Longtine.213-346.jpg

  • We see a band at 2.5kb as expected, but we also have a funky band between 1kb and 850bp. Will gel purify 2.5kB band, rerun PCR with some of the purified product, transform with the rest.
  • Funky band seems to indicate mispriming, although we're not quite sure of what (probably a fragment of the 2.5kb Longtine part?)
  • Will try raising the temperature of PCR, using a MgCl gradient (0, 1, 2, 4) to see if different conditions will select for 2.5kb band.
Magnesium gradient
0: 4.5μL ddH2O, 0μL 20mM MgCl
1: 3.5μL ddH2O, 1μL 20mM MgCl
2: 2.5μL ddH2O, 2μL 20mM MgCl
4: 3μL   ddH2O, 2μL 20mM MgCl
Total: =4.5μL H20/MgCl solution/20μL reaction
  • Again if this doesn't make sense it's because we haven't yet realized our recipe is weird. Please see protocols and/or notebook entry for 7/1 for the correct PCR reaction recipe
  • Run PCR with elongation temperature raised to 62C
  • -DNA and -pol controls (only one set for entire PCR)

Gel Purification

  • Gel purification (Damon) of suspected product band from PCR on 6/29 (See protocols for exact procedure)
  • Gel cast on two teeth of the comb taped together to create bigger wells, also under aluminum foil
  • Used 5mL SybrSafe in 100mL agarose (1.2%) because we ran out
  • Preheated water ath to 50°C.
  • Concentrations of gel-purified products (using Qubit fluorometer)
035 = 11.9 μg/mL 
139 = 8.72μg/mL 
049 = 3.1 μg/mL 
186 = 11.56μg/mL 
213 = 8.12 μg/mL 
236 = 25.8 μg/mL
346 = 10.22μg/mL
  • Definitely enough for PCR and/or transformations
  • Will do PCR on 7/1