Biomod/2013/Waterloo: Difference between revisions
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#Add 2.5 uL of 100 mM EDTA to the PCR tube | #Add 2.5 uL of 100 mM EDTA to the PCR tube | ||
#Vortex the PCR tube for 30 seconds | #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 | 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 following table: |
Revision as of 17:22, 25 October 2013
Assembly of the Walker
Video
Kristopher hurry up.
Required Materials
- 1M Tris-HCl, pH 7.5
- 0.5M Acetic Acid
- 100mM EDTA
- 100mM Magnesium Acetate
- DNA Working Stocks (~1uM): Walker-{1-7}
- 1x 200uL PCR tube
Procedure
- 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 following table: