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==Procedure== *Prepare more E.coli gDNA MDA amplicons for testing. **Preparing UV-ed ALS, NS buffer 50uL-MDA No-SYBR recipe: 1 rxn 4.5rxn --------------------------------------------- RT-PCR grade H2O 29.0 130.5 10x RepliPhi phi-29 buffer 5.0 22.5 1mM N6 primer 2.5 11.25 RepliPhi Phi-29 (100U/ul) 2.5 11.25 dNTP (25mM) 2.0 9.0 ---------------------------------------------- 41.0 184.5/4.5=41 3uL ALS buffer + 3uL E.coli gDNA (1ng/uL) + 3uL NS buffer Transfer 41uL master mix into lysed template. Incubate at 30C for 8hr. Inactivate by 85C 3min. *Pool the four tubes of MDA amplicons into one and quantify the yield using QUBIT-HS dsDNA **Result: 666ng/uL in 200uL. *Take 1ug Amplicon and dilute into 100uL H2O in a 1.5mL-tube for shearing. ===Amplicon shearing test=== *Directly shear the raw MDA amplicons (1ug) without purification. *Following operation manual from 09/09/'10 with following changes on settings. **Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(10min). **Take out 20uL (200ng) sample for validation every 2min.
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