Chris:LabNotes/FateMapping/Calendar/2014/2014-10-30: Difference between revisions

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==Background==
==Background==
*The previous two tests didn't seem to have worked (CircLigase didn't seem to have circularized either the sample or the positive control)
*The previous two tests didn't seem to have worked (CircLigase didn't seem to have circularized either the sample or the positive control)
*This time, I'll repeat the experiment but only use CircLigase II
*This time, I'll repeat the experiment but only use CircLigase II and making sure that all the reagent concentrations are correct in the rxn mixes (such as adding the appropriate amount of betain to the CircLigaseII rxn)
 
==Procedure==
==Procedure==
*Below are the reactions I want to do for this experiment:
*Below are the reactions I want to do for this experiment:

Revision as of 22:18, 5 November 2014

Bisulfite Conversion Test 3

Background

  • The previous two tests didn't seem to have worked (CircLigase didn't seem to have circularized either the sample or the positive control)
  • This time, I'll repeat the experiment but only use CircLigase II and making sure that all the reagent concentrations are correct in the rxn mixes (such as adding the appropriate amount of betain to the CircLigaseII rxn)

Procedure

  • Below are the reactions I want to do for this experiment:
Rxn 1 (gDNA)    Bisulfite Conversion
Rxn 2 (gDNA)    Bisulfite Conversion -> T4 PNK -> CircLigase II -> Exo I/III
Rxn 3 (NTC)     Bisulfite Conversion
Rxn 4 (NTC)     Bisulfite Conversion -> T4 PNK -> CircLigase II -> Exo I/III

EZ DNA Methlyation-Direct

  • Use human gDNA (110ng/ul) for template for rxn's 1->4 (use 3.64ul to get 400ng of gDNA)
  • Follow online published protocol (make sure to prep using specifications for smaller D5020 kit)
  • Final elution = 10ul
  • Note: Can quantify bisulfite conversion recovery by using Qubit ssDNA assay