Matt:LabNotes/2014-2-10

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Quantify ppMALAT1 in vitro Capture from 2/6/2014[edit]

Samples

  • 8 PCR tubes of 35ul from 2/6/2014
    • Actual volume ~30ul due to evaporation

First trial of quantifying[edit]

Procedure

  • Took the first and last tube
  • Brought each sample to 50ul with 20ul H2O
  • Used Zymo ssDNA/RNA Clean & Concentrate Kit following standard procedure
  • Eluted with 15ul H2O and quantified each with 1ul using Qubit ssDNA

Results

  • Starting mass of linear ppMALAT1 in each tube: 3ul * 1434 ng/ul = 4.302 ug
  • Tube1: 68.8 ng/ul
    • 15ul * 68.8 ng/ul = 1032 ng = 1.032 ug
    • Yield = 1.032/4.302 = 24%
    • 1.032 ug * (umole/28019.4ug) = 0.0000368 umole = 36.83 pmole
    • 36.83 pmole/15 ul = 2.46 pmole/ul = 2.46 uM
  • Tube2: 75.3 ng/ul
    • 15ul * 75.3 ng/ul = 1129.5 ng = 1.1295 ug
    • Yield = 1.1295/4.302 = 26%
    • same calculation as above -> 2.69 uM

Run Gel[edit]

  • Run denaturing TBU gel to check circularization
    • Lane 1: 0.5ul Low Mass ladder
    • Lane 3: 2ul 2uM ppMALAT1 linear ssDNA
    • Lane 4: 2ul 2uM ppMALAT1 linear ssDNA after Zymo ssDNA purification (from PosCtrl)
    • Lane 5: 2ul of circularized ppMALAT1 (from 1000ng capture)
    • Lane 6: 0.5ul of circularized ppMALAT1 from Tube 1 (68.8ng/ul)

File:2014-02-10 ppMALAT1 Capture Check.jpg

Purifying the rest[edit]

Procedure

  • Raised the volumes of the other 6 tubes to 50ul with H2O
  • Combined them into 3 tubes of 100ul
  • Used Zymo ssDNA/RNA Clean & Concentrate Kit
  • Eluted with 15ul each -> 45ul total