EricChu:LabNotesMDA/2014-1-27

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Redo Real Time MDA to Check Protease in MDA in PCR tubes[edit]

  • From the previous experiment, all conditions involving protease were either poorly amplified or non-amplified. Also excess (20% more) neutralization solution (HCl+TrisHCl) did not affect MDA reaction.
  • This experiment tested the adverse effect of heat, protease or protease buffer, with heat inactivation.
  • This experiment also tested the excess of denaturation buffer in MDA in 20ul volume.
  • Since genomic DNA behaved similarly to cells, 60pg/ul human genomic DNA was used.
  • Here are the tested conditions:
  1. with protease buffer. no heat.
  2. with protease buffer. 40C for 15min.
  3. without protease buffer. 40C for 15min.
  4. without protease buffer. normal denaturation buffer.
  5. without protease buffer. 10% more denaturation buffer.
  6. without protease buffer. 20% more denaturation buffer.
  7. with protease buffer. 50C for 15min.
  8. with protease. 40C 15min then 70C 15min (deactivation).
  9. with deactivated protease (70C 15min in PCR tube).
  10. with protease. 40C 15min. no deactivation (duplicating previous result).

File:01272014realtimeMDA.jpg

Result[edit]

  • Protease needed deactivation by heat for a successful MDA run.
  • KOH did not deactivate the protease.
  • Amount of KOH was very significant. MDA reaction was very sensitive to KOH concentration.