AlanFung:LabNotes/CTCF/2011-8-26

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Revision as of 06:23, 27 August 2011 by >Alan6017518 (→‎Protocols Today)
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Low-input Bisulfite Conversion and MDA Nextera library

Protocols Today

  • Start with four tubes: 314 cells, 157 cells x 2, NTC all go through bisulfite conversion
  • Make 1571 cell/ul (original tube)
  • Make 314 cells/ul (dilute 10ul cells in 40 ul ddh2o)
  • Make 157cells/ul (dilute 10ul cells in 90 ul ddh2o)

Cell Lysis

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
  • Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Cells are now lysed

Make New BSA-Water solution

  • Stock solution is at 10mg/ml, need to dilute to 0.5mg/ml
  • Take 10ul stock BSA dilute with 190ul nuclease free water

Bisulfite Conversion - Imprint Kit

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 21 uL 0.5 mg/mL BSA and
  • Add1 uL Imprint Balance solution to each sample;
  • incubate at 37 C for 10 minutes
  • Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
  • Perform Column Purification of each sample:
  • Elute into 11uL Elution Buffer

Continue on 8/27

  • Denature all samples at 95C for 30sec and quickly transfer to cooler rack