Revision as of 08:38, 26 October 2013

Video

Remove DNA working stocks from the freezer and allow them to thaw
Prepare the buffer:
Add 49.5 uL of milli-Q water to the PCR tube
Add 12.5 uL (6+6.5uL) of 100mM Magnesium Acetate to the PCR tube
Add 4 uL of 1M Tris-HCl to the PCR tube
Add 4 uL of 500 mM Acetic Acid to the PCR tube
Add 2.5 uL of 100 mM EDTA to the PCR tube
Vortex the PCR tube for 30 seconds
Determine A260 of each strand using the spectrophotometer using Oligocalc (http://www.basic.northwestern.edu/biotools/OligoCalc.html) and the sequence of the strand, determine the concentration of the strand. The concentration of each strand in the PCR tube must be 50nM. Add a volume of the DNA working stock to the PCR tube equal to the table below.
Close the PCR tube, tap down any droplets that may be on the sides of the container and vortex for one minute
Open the PCR machine and place the PCR tube inside any of the compartments
Close the PCR machine, go to the computer and run qCycle.exe
Press the Load/Edit button and open the appropriate walker synthesis script, depending on the cooling duration requirements for that trial
Press Start Program
Enter 100 uL into the text box when prompted for the solution volume
Allow the PCR machine to run. This process will take a variable amount of time, depending on the length of the cooling process.

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