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='''MDA on sorted human skin microbiome sample by SIO'''= ===Background=== *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=== *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=== [[File:031512-MDA-on-HSM_SIO.jpg|400px]] *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.
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