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EricChu:LabNotesMDA/2014-1-27
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==Redo Real Time MDA to Check Protease in MDA in PCR tubes== * 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: # with protease buffer. no heat. # with protease buffer. 40C for 15min. # without protease buffer. 40C for 15min. # without protease buffer. normal denaturation buffer. # without protease buffer. 10% more denaturation buffer. # without protease buffer. 20% more denaturation buffer. # with protease buffer. 50C for 15min. # with protease. 40C 15min then 70C 15min (deactivation). # with deactivated protease (70C 15min in PCR tube). # with protease. 40C 15min. no deactivation (duplicating previous result). [[File:01272014realtimeMDA.jpg]] ==Result== * 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.
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