Biomod/2013/Waterloo

Required Materials

1. 1M Tris-HCl, pH 7.5
2. 0.5M Acetic Acid
3. 100mM EDTA
4. 100mM Magnesium Acetate
5. DNA Working Stocks (~1uM): Walker-{1-7}
6. 1x 200uL PCR tube

Procedure

1. Remove DNA working stocks from the freezer and allow them to thaw
2. Prepare the buffer:
3. Add 49.5 uL of milli-Q water to the PCR tube
4. Add 12.5 uL (6+6.5uL) of 100mM Magnesium Acetate to the PCR tube
5. Add 4 uL of 1M Tris-HCl to the PCR tube
6. Add 4 uL of 500 mM Acetic Acid to the PCR tube
7. Add 2.5 uL of 100 mM EDTA to the PCR tube
8. Vortex the PCR tube for 30 seconds
9. 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.
10. Close the PCR tube, tap down any droplets that may be on the sides of the container and vortex for one minute
11. Open the PCR machine and place the PCR tube inside any of the compartments
12. Close the PCR machine, go to the computer and run qCycle.exe
13. Press the Load/Edit button and open the appropriate walker synthesis script, depending on the cooling duration requirements for that trial
14. Press Start Program
15. Enter 100 uL into the text box when prompted for the solution volume
16. Allow the PCR machine to run. This process will take a variable amount of time, depending on the length of the cooling process.

Strand-ID	Volume (μL)
Walker 1	3.9
Walker 2	5.3
Walker 3	4.3
Walker 4	3.3
Walker 5	3.9
Walker 6	3.9
Walker 6	2.9

Assembly of the Cassettes

The cassettes are much more complex than the walker, as they are made up of many more strands.

Only one cassette should be assembled at a time within each PCR tube.

Required Materials

• 1M Tris-HCl, pH 7.5
• 0.5M Acetic Acid
• 100mM EDTA
• 100mM Magnesium Acetate
• DNA Working Stocks (~1uM)
• 1x 200uL PCR tube

Procedure

1. Remove DNA working stocks from the freezer and allow them to thaw
2. Prepare the buffer:
3. Add milli-Q water to the PCR tube:
4. 11.2 uL for Cassette 1. 8.1 uL for Cassette 2. 10.2 uL for Cassette 3
5. Add 25 uL of 100mM Magnesium Acetate to the PCR tube
6. Add 8 uL of 1M Tris-HCl to the PCR tube
7. Add 8 uL of 500 mM Acetic Acid to the PCR tube
8. Add 5 uL of 100 mM EDTA to the PCR tube
9. Vortex the PCR tube for 30 seconds
10. Determine A260 of each strand using the spectrophotometer.
11. Using Oligocalc (http://www.basic.northwestern.edu/biotools/OligoCalc.html) and the sequence of the strand found, 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 previously calculated values.
12. Close the PCR tube and vortex for one minute
13. Open the PCR machine and place the PCR tube inside any of the compartments
14. Close the PCR machine, go to the computer and run qCycle.exe
15. Press the Load/Edit button and open the appropriate cassette synthesis script, depending on the cooling duration requirements for that trial
16. Press Start Program
17. Enter 150 uL into the text box when prompted for the solution volume
18. Allow the PCR machine to run. This process will a variable amount of time, depending on the requirements for that trial