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.