EricChu:LabNotesMDA/2014-1-21
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Running Real-time MDA in PCR tubes to test different MDA conditions[edit]
- Use both PGP1 cells and genomic human DNA
- 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubated for 1min in room temp.
- 1ul 400mM NS was added. Incubated for 1min in room temp.
- Added 1ul of (i) water (no protease) or (ii) 24mM protease and incubate at 40C or 50C for 15min
- Added 1ul of 250mM KOH (final 50mM) or 100mM KOH (final 20mM)
- Added 5ul of 50mM HCl or 20mM HCl (according to the previous step). Also added 5.5ul HCl (10% extra) and 6ul HCl (20% extra) to the test.
- Took 1ul out of the 10ul from above (equivalent to 10 cells) and added to 40ul MDA mix with SYBR Green.
Summary of Different Conditions[edit]
- DNA: 10 cells or 60pg human genomic DNA
- Cell lysis and DNA denaturation were identical in all cases.
- Neutralization: identical in all cases.
- Protease: (i) absent (ii) 40C 15min or (iii) 50C 15min
- DNA denaturation: final 50mM KOH or final 20mM KOH
- Neutralization: (i) HCl equal molar to KOH, (ii) 10% more HCl or (iii) 20% more HCl
- MDA: all identical conditions
Recipes[edit]
CoRE Mastermix[edit]
- 4ul 10x buffer
- 4ul 8% Tween20
- 10ul N6 primer
- 2ul dNTP
- 2.5ul dUTP (1mM)
- 4ul 10x SYBR Green
- 0.4ul Phi29 (high)
- 13.1ul water
250mM (or 100mM) KOH for 50mM (or 20mM) final KOH[edit]
- 5M KOH 5ul (or 2ul)
- 2M DTT 5ul
- 0.5M EDTA 0.1ul
- 8% Tween20 12.5ul
- water 77.4ul (or 80.4ul)
Result[edit]
- All conditions were shown in the following diagram.
File:01232014allconditions.jpg
- All conditions without protease were amplified similarly. No visible effect was seen with 10% or 20% more HCl. Therefore the TrisHCl buffer was working pretty nicely in these case.
- Samples with protease did not amplify at all or did not amplify well. Something was inhibiting the MDA reaction.