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
  1. 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubated for 1min in room temp.
  2. 1ul 400mM NS was added. Incubated for 1min in room temp.
  3. Added 1ul of (i) water (no protease) or (ii) 24mM protease and incubate at 40C or 50C for 15min
  4. Added 1ul of 250mM KOH (final 50mM) or 100mM KOH (final 20mM)
  5. 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.
  6. 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]

  1. DNA: 10 cells or 60pg human genomic DNA
  2. Cell lysis and DNA denaturation were identical in all cases.
  3. Neutralization: identical in all cases.
  4. Protease: (i) absent (ii) 40C 15min or (iii) 50C 15min
  5. DNA denaturation: final 50mM KOH or final 20mM KOH
  6. Neutralization: (i) HCl equal molar to KOH, (ii) 10% more HCl or (iii) 20% more HCl
  7. MDA: all identical conditions

Recipes[edit]

CoRE Mastermix[edit]

  1. 4ul 10x buffer
  2. 4ul 8% Tween20
  3. 10ul N6 primer
  4. 2ul dNTP
  5. 2.5ul dUTP (1mM)
  6. 4ul 10x SYBR Green
  7. 0.4ul Phi29 (high)
  8. 13.1ul water

250mM (or 100mM) KOH for 50mM (or 20mM) final KOH[edit]

  1. 5M KOH 5ul (or 2ul)
  2. 2M DTT 5ul
  3. 0.5M EDTA 0.1ul
  4. 8% Tween20 12.5ul
  5. 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.

File:01232014noprotease.jpg

  • Samples with protease did not amplify at all or did not amplify well. Something was inhibiting the MDA reaction.

File:01232014allprotease.jpg