AlanFung:LabNotes/CTCF/2011-9-20: Difference between revisions

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* We will test the pH of the NaOH with litmus paper Range 0-14 (Ordered placed on 9/20)  
* We will test the pH of the NaOH with litmus paper Range 0-14 (Ordered placed on 9/20)  
*Add 1ul NaOH to 24ul nuclease free water, test pH with litmus paper (Increase amount of NaOH if the pH is not high enough)
*Add 1ul NaOH to 24ul nuclease free water, test pH with litmus paper (Increase amount of NaOH if the pH is not high enough)
*Both pH are at 14
*Both pH are at close to 14

Revision as of 21:45, 26 September 2011

Cell Lysis

  • Instead of using protease and NP40 lysis buffer, we will use NaOH for cell lysing, ideally NaOH should be made fresh as it will absorb carbon dioxide and form sodium bicarbonate which does not lyse cells
  • In the original imprint protocol, we adjust the total volume of DNA sample to 24ul with water and add 1ul NaOH (Balance Solution), vortex and incubate sample at 37C for 10min, in order to denature the DNA samples
  • Protocol for cell lyses found online uses 200mM NaOH/1%SDS (Where the NaOH breaks down the cell wall and denatures the DNA, while SDS helps by making the cell wall soluble and denatures the protein)
  • The question now is what is the concentration and volume needed to lyse the cells, we have no information about the concentration of the provided NaOH, I will try to use it for cell lyses with 1% of SDS.
  • Prepare Stock Balance Solution with 1% SDS
  • After adding NaOH gentle mixing is required so the DNA is not damaged. The DNA will have various (relatively large) proteins bound and anything more than gentle inversion or rolling the falcon can shear the DNA

Prepare 10% SDS

  • Add 1g of SDS in 10ml of solvent
  • Dilute 10% SDS to 1% SDS
  • Add 0.1ul 10% SDS to 0.9ul Balance solution (NaOH)

Cell Lysis

  • I would like to do an alkaline cell lysis with NaOH, after doing some research the required amount of OH- has to be at least 20mM NaOH in PBS (pH 11.7) for quick cell lysis
  • We will test the pH of the NaOH with litmus paper Range 0-14 (Ordered placed on 9/20)
  • Add 1ul NaOH to 24ul nuclease free water, test pH with litmus paper (Increase amount of NaOH if the pH is not high enough)
  • Both pH are at close to 14