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EricChu:LabNotesMDA/2014-1-21
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==Running Real-time MDA in PCR tubes to test different MDA conditions == * 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== # 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== ===CoRE Mastermix=== # 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=== # 5M KOH 5ul (or 2ul) # 2M DTT 5ul # 0.5M EDTA 0.1ul # 8% Tween20 12.5ul # water 77.4ul (or 80.4ul) ==Result== * All conditions were shown in the following diagram. [[File:01232014allconditions.jpg|500px]] * 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. [[File:01232014noprotease.jpg|500px]] * Samples with protease did not amplify at all or did not amplify well. Something was inhibiting the MDA reaction. [[File:01232014allprotease.jpg|500px]]
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