Biomod/2013/Waterloo: Difference between revisions
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= Video= | = Video= | ||
[[Movie:http://www.youtube.com/watch?v=a7fymvFQ0CQ]] | |||
Kristopher hurry up. | Kristopher hurry up. | ||
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{| class="wikitable" | {| class="wikitable" | ||
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! | ! Strand-ID !! Volume (μL) | ||
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| Walker 1 || 3.9 | | Walker 1 || 3.9 | ||
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| Walker 6 || 3.9 | | Walker 6 || 3.9 | ||
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| Walker 6 || 2.9 | |||
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Revision as of 17:34, 25 October 2013
Video
Movie:http://www.youtube.com/watch?v=a7fymvFQ0CQ 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:
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 |