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==Planning== * Sequential MDA on C1 with PEO1 * July 2, 2015 * Experiment ID: AR150702_C1 {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''(bases)''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | haploid length|| 3,100,000,000 |||||| |- | diploid length|| 6,200,000,000 |||||| |- | denature length|| 12,400,000,000 |||||| |- | |||||||| |- | phi29 mean processivity|| 70,000 |||||| |- | N9 combinations|| 262,144 ||N6|||| |- | % annealed template at 30 C||98%||2%|||| |- | |||||||| |- | desired gain||100||1000|||| |- | space between primers|| 691 || 61 |||| |- | # N9s to cover every base|| 1,377,777,778 |||||| |- | # N9s for spacing|| 1,968,254 |||||| |- | mols of N9 desired||3.27E-18|||||| |- | |||||||| |- | volume of first MDA (nL)|| 31.5 |||||| |- | volume of first MDA (L)||3.15E-08|||||| |- | concentration of N9 (M)||1.038E-10|||||| |- | concentration of N9 (nM)|| 0.1038 ||1|||| |- | fold change vs 50 uM|| 481,721 |||||| |- | |||||||| |- | primers (uM)||50|||||| |- | primers (M)||0.00005|||||| |- | |||||||| |- | ||C1 31 nL||Microwell 12 nL||volume (L)||1.20E-08 |- | dNTPs (mM)||4.00E-01|||||| |- | dNTPs (M)||4.00E-04|||||| |- | mol dNTPs||1.26E-11|||||| |- | # dNTPs||7.59E+12|||||| |- | mass/dNTPS incorporated (g/mol)||3.30E+02|||||| |- | mass of dntps (g)||4.16E-09|||||| |- | mass of dntps (pg)||4.16E+03|||||| |- | mass of single genome (pg)||6.6|||||| |- | gain|| 630 || 240 |||| |- | |} ===Protocol=== ====C1 setup==== [[File:AR150702_reagents-page-001.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-001.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-002.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-003.jpg|600px]] [[File:AR150702_thermalProfile.jpg|600px]] ====Off chip==== Prep * Add 1.2 uL 50X SYBR Green I to 120 uL NPM Nextera XT *Same as standard protocol, but add 15.5 uL NPM/SYBR Green I mix and run on qPCR machine (final volume 50.5 uL) 0.1X Tn5-059 *5 uL total **3 ul template **1 ul SSB **1 ul 1X Tn5-059 *Incubate 55 C for 5 min, transfer to cold block *Add 1 ul 0.2 mg/ml Qiagen Protease *Incubate 50 C for 15 min, followed by heat inactivation at 70 C for 20 min *Add 19 uL H2O to bring volume up from 6 uL to 25 uL *Add 5 uL each index (N7xx/S5xx) *Add 15.5 uL NPM/SYBR Green I mix qPCR protocol *72 C for 3 min *95 C for 1 min *25 cycles (but prepare to stop early) **95 C for 15 sec **55 C for 30 sec **72 C for 30 sec, acquisition **72 C for 15 sec *72 C for 5 min
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