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=== <span style="color:blue"><u>Part 2: DNA extraction from plasma samples</u></span> === * There are <u>three tubes</u> of plasma for each sample. Dr. Zhang suggested to use 2 tubes for DNA extraction. * I measured volume of two tube serum during DNA extraction and adjust volume to 600ul with 1X PBS as ChargeSwitch kit required different volume of reagent for different sample volume. * Note that the measurement could be +-5ul error since serum is viscous and I needed to adjust the pipette volume after aspiration to measure the volume * I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3- 11#3._ChargeSwitch.C2.AE_gDNA_1_ml_Serum_kit]] for <u>700ul</u> sample volume :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Plasma Volumn (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''PBS to 700ul (ul)''' |- | UCSD-006.1||6-P-1||430.00||270.00 |- | UCSD-006.2||6-P-2||540.00||160.00 |- | UCSD-006.3||6-P-3||220.00||480.00 |- | UCSD-006.4||6-P-4||550.00||150.00 |- | UCSD-006.5||6-P-5||665.00||35.00 |- | UCSD-006.6||6-P-6||650.00||50.00 |- | UCSD-006.7||6-P-7||700.00||0.00 |- | UCSD-006.8||6-P-8||690.00||10.00 |- | UCSD-006.9||6-P-9||520.00||180.00 |- | UCSD-006.10||6-P-10||700.00||0.00 |} : '''''<span style="color:crimson"><u>Prep (for 700ul sample volume)</u></span>'''''<br> :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="width:120px;background:#f0f0f0;"|'''Sample ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Plasma Volumn (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''PBS to 500ul (ul)''' |- | UCSD-007.1||7-P-1||475.00||25.00 |- | UCSD-007.2||7-P-2||350.00||150.00 |- | UCSD-007.3||7-P-3||400.00||100.00 |- | UCSD-007.4||7-P-4||325.00||175.00 |- | UCSD-007.5||7-P-5||440.00||60.00 |- | UCSD-007.6||7-P-6||345.00||155.00 |- | UCSD-007.7||7-P-7||320.00||180.00 |- | UCSD-007.8||7-P-8||303.00||197.00 |- | UCSD-007.9||7-P-9||330.00||170.00 |- | UCSD-007.10||7-P-10||500.00||0.00 |} '''''<span style="color:crimson"><u>Prep (for 500ul sample volume)</u></span>'''''<br> * Eluted 30ul with Elution Buffer * Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution) ==== Qubit dsDNA HS assay result ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:70px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:70px;background:#f0f0f0;"|'''Sample Conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:70px;background:#f0f0f0;"|'''Yield (ng)''' |- | 6-P-1||2||ng/mL||2||100||0.200||30.00||6.00 |- | 6-P-2||11||ng/mL||2||100||1.100||30.00||33.00 |- | 6-P-3||15.7||ng/mL||2||100||1.570||30.00||47.10 |- | 6-P-4||8.89||ng/mL||2||100||0.889||30.00||26.67 |- | 6-P-5||4.87||ng/mL||2||100||0.487||30.00||14.61 |- | 6-P-6||2.49||ng/mL||2||100||0.249||30.00||7.47 |- | 6-P-7||2.47||ng/mL||2||100||0.247||30.00||7.41 |- | 6-P-8||2.26||ng/mL||2||100||0.226||30.00||6.78 |- | 6-P-9||4.08||ng/mL||2||100||0.408||30.00||12.24 |- | 6-P-10||3.33||ng/mL||2||100||0.333||30.00||9.99 |} {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:70px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:70px;background:#f0f0f0;"|'''Sample Conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:70px;background:#f0f0f0;"|'''Yield (ng)''' |- | 7-P-1||6.92||ng/mL||2||100||0.692||30.00||20.76 |- | 7-P-2||11.4||ng/mL||2||100||1.140||30.00||34.20 |- | 7-P-3||2.08||ng/mL||2||100||0.208||30.00||6.24 |- | 7-P-4||2.02||ng/mL||2||100||0.202||30.00||6.06 |- | 7-P-5||11.1||ng/mL||2||100||1.110||30.00||33.30 |- | 7-P-6||2.33||ng/mL||2||100||0.233||30.00||6.99 |- | 7-P-7||2.29||ng/mL||2||100||0.229||30.00||6.87 |- | 7-P-8||1.67||ng/mL||2||100||0.167||30.00||5.01 |- | 7-P-9||2.31||ng/mL||2||100||0.231||30.00||6.93 |- | 7-P-10||2.03||ng/mL||2||100||0.203||30.00||6.09 |} ==== Normalize DNA yield to 1mL plasma volume ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Serum volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Normalized yield to 1mL (ng)''' |- | 6-P-1||6.00||430.00||13.95 |- | 6-P-2||33.00||540.00||61.11 |- | 6-P-3||47.10||220.00||214.09 |- | 6-P-4||26.67||550.00||48.49 |- | 6-P-5||14.61||665.00||21.97 |- | 6-P-6||7.47||650.00||11.49 |- | 6-P-7||7.41||700.00||10.59 |- | 6-P-8||6.78||690.00||9.83 |- | 6-P-9||12.24||520.00||23.54 |- | 6-P-10||9.99||700.00||14.27 |} '''UCSD-006 Plasma original color'''<br> [[File:UCSD-006-plasma_1-10_1_e.jpg|500px]] * Based on plasma color, 6-P_3 sample had very high DNA amount due to cell lysis {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Serum volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Normalized yield to 1mL (ng)''' |- | 7-P-1||20.76||475.00||43.71 |- | 7-P-2||34.20||350.00||97.71 |- | 7-P-3||6.24||400.00||15.60 |- | 7-P-4||6.06||325.00||18.65 |- | 7-P-5||33.30||440.00||75.68 |- | 7-P-6||6.99||345.00||20.26 |- | 7-P-7||6.87||320.00||21.47 |- | 7-P-8||5.01||303.00||16.53 |- | 7-P-9||6.93||330.00||21.00 |- | 7-P-10||6.09||500.00||12.18 |} '''UCSD-007 Plasma original color'''<br> [[File:UCSD-007-plasma_1-10_1.jpg|500px]]
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