EricChu:LabNotesMDA/2014-1-27: Difference between revisions
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==Redo Real Time MDA to Check Protease in MDA in PCR tubes== | ==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. | * 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 protease or protease buffer, with heat inactivation. | * 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. | * 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. | * Since genomic DNA behaved similarly to cells, 60pg/ul human genomic DNA was used. | ||
* Here are the tested conditions: | * 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. |
Latest revision as of 23:23, 5 February 2014
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:
- 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).
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.