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===Procedure=== * Aliquot before adding 1ul Ptnase (1:100d) * Test Trp-R * Test Trp-B/O and Trp-P/P primer sets * Test different Ta {| {{table}} | align="center" style="background:#f0f0f0;"|'''Cell lysis and aliquot''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Notes:''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||1 tube with 1ul of 10 cells/ul||||Try to reduce physical force and lysis time|| |- | ||add 60ul 1X lysis buffer, mixed||||No pipetting, gently stir by P20 tip|| |- | ||37C, 5-10min|||||| |- | |- | Protease treatment|||||||| |- | ||add 0.6ul protease ||||Aliquot before protease addition || |- | ||mix and aliquot to 24 tubes||||Aliquot by using P20 tip || |- | ||50C, 15min||||and try to use contineous gun|| |- | ||70C, 15min|||||| |- | |- | Tagmentation|||||||| |- | ||DNA||2||Using Trp-Roche|| |- | ||5X LMWbuffer||1||Trp-I as ctrl|| |- | ||1:50 dilted enzyme||0.4|||| |- | ||H2O||1.6|||| |- | ||total||5|||| |- | ||55C, 5min|||||| |- | |- | Protease treatment|||||||| |- | ||dilute protease to 1:500|||||| |- | ||add 1ul to each tube|||||| |- | ||50C, 10min|||||| |- | ||70C, 20min|||||| |- | |- | 1st round of PCR||||||Using Trp-B/O primer set (R1/R2)|| |- | ||DNA||6||Using Trp-P/P primer set|| |- | ||Kapa fast mix||15||Test different Ta|| |- | ||Orange (10uM)||0.24||55C, 52C, 50C, 48C|| |- | ||blue (10uM)||0.24|||| |- | ||H2O||8.52|||| |- | ||Total||30|||| |- | ||72C, 3min|||||| |- | ||95C 30sec, [95C 10sec; 58C 30sec; 72C 2min] X 20||||||58C |- | ||72C, 3min|||||| |}
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