Matt:LabNotes/2014-2-5

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Quantifying Pre-circularized ppMALAT1[edit]

Samples

  • 5ul of 10ng capture product: stored in 4C
  • 9ul of 1000ng capture product: stored in -20C
  • Positive control: 20ul of 2uM ppMALAT1 (linear ssDNA) = 40pmole x (92*303.7 + 79) pg/pmole = 1.12ug
  • Negative control: 25ul of dsDNA PCR product from Noi (~1ug)

Procedure[edit]

Purify circular ssDNA with Zymo Kit

  1. Bring each sample to 60ul total with H2O
  2. Use Zymo ssDNA/RNA Clean & Concentrate Kit following default protocol
  3. Elute with 15ul H2O

Quantify with Qubit ssDNA Assay

  1. Buffer mix: 6ul reagent + 1194ul buffer = 1200ul
  2. Add 10ul of each standard control to 190ul buffer mix
  3. Add 1ul of each sample to 199ul buffer mix

Results[edit]

  • 10ng capture product: too low to detect
    • minimum detectable is 50pg/ul = 50ng/ml
  • 1000ng capture product: 15.1ug/ml
  • PosCtrl: 16.0ug/ml
  • NegCtrl: too low to detect
    • Repeated qubit on 2/10/14 with 10ul of sample to find 30ng/ml

Calculations[edit]

  • PosCtrl: 16 ug/ml = 16 ng/ul
    • 15ul * 16 ng/ul = 240ng
    • 240ng/1.12ug = 21.4% yield
  • 1000ng: 15.1 ug/ml = 15.1 ng/ul
    • 15ul * 15.1 ng/ul = 226.5ng purified circular ssDNA
    • Starting amount of ppMALAT1 used in capture reaction: 2ul * 1434 ng/ul = 2.868 ug (from Alan's notes)
    • Fraction used for purification/quantification: 9/15 * 2.868 ug = 1.7208 ug
    • 226.5ng/1.7208ug = 13.16% yield (includes padlock capture step)
      • Assuming circular ssDNA has same yield during purification as linear ssDNA (PosCtrl), then 61.5% of ppMALAT1 is circularized
    • 226.5 ng * (nmole/28019.4ng) = 0.00808 nmole = 8.08 pmole
    • 8.08 pmole/15 ul = 0.539 pmole/ul = 0.539 uM
      • If none was used for PCR: 0.539 uM * 15/9 = 0.9uM

How much pre-circularized ppMALAT1 do we need for positive control?

  • 1uM seems pretty reasonable considering that is about what we use for fluorescent-dye probes to detect primary rolonies
    • Theoretically that mean it's more than the number of primary rolonies and even if we can only fix 1/10th of them that's enough for positive control
  • Need atleast 150ul so we can do 3 wells with positive control