IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/06/30: Difference between revisions
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* Overnight culture OD=1.754 (wavelength 660nm) | * Overnight culture OD=1.754 (wavelength 660nm) | ||
10mL of YPD media+.2μL overnight culture | 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 | |||
* Gel purification (Damon) of suspected product band from PCR on 6/29 | [[Image: 2009-06-30.Longtine0.35-186.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 | * 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 | * Used 5mL SybrSafe in 100mL agarose (1.2%) because we ran out | ||
* Preheated water ath to 50°C. | * Preheated water ath to 50°C. | ||
* Concentrations of gel-purified products | * Concentrations of gel-purified products (using Qubit fluorometer) | ||
035 = 11.9 μg/mL | 035 = 11.9 μg/mL | ||
139 = 8.72μg/mL | 139 = 8.72μg/mL | ||
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236 = 25.8 μg/mL | 236 = 25.8 μg/mL | ||
346 = 10.22μg/mL | 346 = 10.22μg/mL | ||
*Definitely enough for PCR and/or transformations | |||
*Will do PCR on 7/1 | |||
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Revision as of 12:58, 6 July 2009
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June 30, 2009
10mL of YPD media+.2μL overnight culture
PCR
Gel 1: 1 kb Ladder, -DNA, -Enz, 035, 139, 049, 186 Gel 2: 1 kb Ladder, -DNA, -Enz, 213, 236, 346
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
Gel Purification
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
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