Sam:LabNotes/Microbiome-new/2012-3-15

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MDA on sorted human skin microbiome sample by SIO[edit]

Background[edit]

  • Test if regular KOH lysis is effective at a single cell level.
    • Confirm Bigelow's experiment results.
    • Evaluate if lysozyme treatment is necessary.
  • Procedures follows original protocol from 09-29-'11 wikinote
  • Will do three strips and realtime monitoring to check if
  • Prepare ALS and NS buffer
    • Original ALS buffer: 200uL H2O + 25uL 1M DTT, 20uL 5M KOH, 5uL 0.5M EDTA-> make 1/3.33d
    • Original NS buffer: use the old one
    • Test the pH value (10uL 1/3.33d ALS and 10uL 1/3.33d NS)
  • UV 15min in the hood and the cross-linker

Set up MDA Master Mix[edit]

  • H20: 0.6 (uL), 10x buffer: 1.0, 1mM N6 primer: 0.5, Phi29 polymerase:0.5, 25mM dNTPs:0.4uL, SYBR-green 0.2X
  • Add 1/3.3d ALS buffer (2uL). Slowly pipetting for 4 times. Incubate at RT for 3min.
  • Add 1/3.3d NS buffer (2uL). Slow pipetting for 4 times.
  • Transfer 3uL complete master mix to each of individual reactions. Mix by (Multi-chan pipettor) repeat pipetting (4X, slowly).
  • Incubate at PCR thermocycler for at 30C for 3hr or longer depends on when the curves raised up.

Results[edit]

File:031512-MDA-on-HSM SIO.jpg
  • Most of wells showed positive amplification, although I can't tell if there are from single cell or contamination.
  • The amplificaion kinetics is different (faster) than my previous experiment, which maybe due to the different qPCR strip tubes were used this time (Eppendorf tube in Bio-Rad Chromo4 machine).
  • Will continue to run 16S PCR to confirm the contented of MDA amplicons.