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Sam:LabNotes/Microbiome-new/2010-6-9 exp2
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='''50uL-MDA 10hr amplification on VA-sorted (EtOH fixed) E.coli (06-09-'10 sorted)'''= ==Background== *Test the reagent cleanse by contamination level test. *Test the MDA amplification on new sorted E.coli (EtOH-fixed) cells. ==Procedures== *Dilute Bacteria gDNA from 1ng/uL to 10pg/uL -> 100fg/uL -> 1 fguL -> 0.01 fg/uL *Step1: Thaw reagents. *Step2: Set up 1.5mL-tubes(x5), PCR-tube caps, and 96-well rack, ALS-NS UV-set (tray + strip tubes) *Step3: Making frest 5M KOH and ALS buffer **5M KOH: Weigh X gram KOH pellet. Add nuclease-free H2O: X/56.11 / 5 * 10^6 (KOH=56.11) **ALS: Mix 200ul nuclease-free H2O, 25uL 1M DTT, 20ul 5M KOH, 5 ul 0.5M EDTA *Step4: Test the pH of ALS + NS (expected to be ~7.5). UV-treatment on ALS and NS in 20uL scale (15min). *Step5: UV-treated the PCR hood (15min) *Step6: Pool the UV-ed ALS and NS. *Step7: Prepare incomplete master mix - 50uL-reaction vol. With SYBR recipe: 1 rxn x (8+5 +2) ---------------------------------- RT-PCR grade H2O 26.5 10x RepliPhi phi-29 buffer 5.0 1mM primer 2.5 2X SYBR Green I 2.5 RepliPhi Phi-29 (100U/ul) 2.5 ----------------------------------- 39.0 25mM dNTP 2.0 --- add dNTP later *Step8: Prepare the sample on rack at RT. *Step9: Set up timer for 5min *Step10: 3uL ALS lysis for 5min. Followed by 3uL NS nutralization. *Step11: Replace the tubes onto cold block. *Step12: Add dNTP into master mix *Step13: Transfer 41uL complete master mix into each reaction. *Step14: Cap the tube caps and seal tightly. *Setp12: Incubate at '''30C for 10hr'''. Followed by 85C 3min inactivation. ==Results== [[File:.sam060910-MDA contamination test.JPG|400px]] [[File:.sam060910-MDA 50uL MDA.JPG|400px]] *The results showed 1~100fg contamination in both my sample and standard curves. **The contamination may exist in the reagents I used.
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