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== <span style="color:blue">Part I: Gel Size Selection </span> == === Step 1. Pool libraries for size selection === {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="188" colspan="4" height="15" | Tube ID: NP-BSPP_V5A_NC.p1-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-1_41 | NC-1 | align="center" | 5.23 | align="center" | 13.39 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-2_42 | NC-2 | align="center" | 5.42 | align="center" | 12.92 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-3_43 | NC-3 | align="center" | 9.44 | align="center" | 7.42 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-5_44 | NC-5 | align="center" | 4.74 | align="center" | 14.77 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-6_45 | NC-6 | align="center" | 5.54 | align="center" | 12.64 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-7_46 | NC-7 | align="center" | 4.44 | align="center" | 15.76 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-8_47 | NC-8 | align="center" | 4.01 | align="center" | 17.46 |- style="background-color:#FF0000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-9_48 | NC-9 | align="center" | 5.49 | align="center" | 12.76 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" | colspan="4" height="15" | Tube ID: NP-BSPP_V5A_NC.p2-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-12_49 | NC-12 | align="center" | 2.41 | align="center" | 29.09 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-13_50 | NC-13 | align="center" | 2.14 | align="center" | 32.66 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-14_51 | NC-14 | align="center" | 2.7 | align="center" | 25.91 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-15_52 | NC-15 | align="center" | 2.65 | align="center" | 26.45 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-16_53 | NC-16 | align="center" | 2.6 | align="center" | 26.95 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-17_54 | NC-17 | align="center" | 3.37 | align="center" | 20.8 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-18_55 | NC-18 | align="center" | 2.26 | align="center" | 31.01 |- style="background-color:#FF6600;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-19_56 | NC-19 | align="center" | 3.55 | align="center" | 19.72 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" | colspan="4" height="15" | Tube ID: NP-BSPP_V5A_NC.p3-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-20_57 | NC-20 | align="center" | 5.5 | align="center" | 18.17 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-21_58 | NC-21 | align="center" | 6.45 | align="center" | 15.52 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-22_59 | NC-22 | align="center" | 6.02 | align="center" | 16.61 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-23_60 | NC-23 | align="center" | 6.1 | align="center" | 16.38 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-25_61 | NC-25 | align="center" | 5.68 | align="center" | 17.61 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-27_62 | NC-27 | align="center" | 5.85 | align="center" | 17.08 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-29_63 | NC-29 | align="center" | 5.82 | align="center" | 17.18 |- style="background-color:#FFFF00;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-NC-30_64 | NC-30 | align="center" | 7.19 | align="center" | 13.9 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" | colspan="4" height="15" | Tube ID: NP-BSPP_V5A_PCP-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-2_65 | PCP-2 | align="center" | 3.06 | align="center" | 22.9 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-3_66 | PCP-3 | align="center" | 2.32 | align="center" | 30.13 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-4_67 | PCP-4 | align="center" | 2.55 | align="center" | 27.46 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-5_68 | PCP-5 | align="center" | 3.56 | align="center" | 19.65 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-6_69 | PCP-6 | align="center" | 1.99 | align="center" | 35.16 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-7_70 | PCP-7 | align="center" | 2.25 | align="center" | 31.16 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-8_71 | PCP-8 | align="center" | 2.07 | align="center" | 33.81 |- style="background-color:#CCFFCC;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-PCP-9_72 | PCP-9 | align="center" | 4.03 | align="center" | 17.36 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" | colspan="4" height="15" | Tube ID: NP-BSPP_V5A_6P-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-1_73 | 6P-1 | align="center" | 4.68 | align="center" | 17.08 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-2_74 | 6P-2 | align="center" | 3.99 | align="center" | 20.05 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-3_75 | 6P-3 | align="center" | 6.79 | align="center" | 11.77 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-4_76 | 6P-4 | align="center" | 4.62 | align="center" | 17.31 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-5_77 | 6P-5 | align="center" | 4.63 | align="center" | 17.26 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-8_78 | 6P-8 | align="center" | 5.58 | align="center" | 14.35 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-9_79 | 6P-9 | align="center" | 3.99 | align="center" | 20.05 |- style="background-color:#008000;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-6P-10_80 | 6P-10 | align="center" | 4.87 | align="center" | 16.42 |- style="font-size:12pt" align="center" | align="center" height="15" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" | colspan="4" height="15" | Tube ID: NP-BSPP_V5A_7P-Dec16 |- style="font-size:12pt" align="center" | height="15" | Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-1_81 | 7P-1 | align="center" | 3.42 | align="center" | 20.48 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-2_82 | 7P-2 | align="center" | 1.6 | align="center" | 43.62 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-3_83 | 7P-3 | align="center" | 3.86 | align="center" | 18.13 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-5_84 | 7P-5 | align="center" | 3.22 | align="center" | 21.74 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-6_85 | 7P-6 | align="center" | 3.25 | align="center" | 21.54 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-7_86 | 7P-7 | align="center" | 4.18 | align="center" | 16.75 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-8_87 | 7P-8 | align="center" | 3.48 | align="center" | 20.13 |- style="background-color:#3366FF;font-size:12pt" align="center" | height="15" | NP-BSPP-V5A-Dec16-7P-10_88 | 7P-10 | align="center" | 3.69 | align="center" | 18.98 |} === Step 2. Add Gel Loading Dye and Load Gel === {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="188" height="15" | Sample ID | width="65" | Total | width="65" | 6X Dye | width="65" | Sum | width="70" | Per Well (4x) |- style="font-size:12pt" |style="background-color:#FF0000" height="15" | Tube ID: NP-BSPP_V5A_NC.p1-Dec16 | align="center" align="center" | 107.12 | align="center" align="center" | 22 | align="center" align="center" | 129.12 | align="center" align="center" | 32.28 |- style="font-size:12pt" |style="background-color:#FF6600" height="15" | Tube ID: NP-BSPP_V5A_NC.p2-Dec16 | align="center" align="center" | 212.59 | align="center" align="center" | 45 | align="center" align="center" | 257.59 | align="center" align="center" | 64.4 |- style="font-size:12pt" |style="background-color:#FFFF00" height="15" | Tube ID: NP-BSPP_V5A_NC.p3-Dec16 | align="center" align="center" | 132.44 | align="center" align="center" | 30 | align="center" align="center" | 162.44 | align="center" align="center" | 27.07 |- style="font-size:12pt" |style="background-color:#CCFFCC" height="15" | Tube ID: NP-BSPP_V5A_PCP-Dec16 | align="center" align="center" | 217.63 | align="center" align="center" | 45 | align="center" align="center" | 262.63 | align="center" align="center" | 65.66 |- style="font-size:12pt" |style="background-color:#008000" height="15" | Tube ID: NP-BSPP_V5A_6P-Dec16 | align="center" align="center" | 134.3 | align="center" align="center" | 30 | align="center" align="center" | 164.3 | align="center" align="center" | 41.07 |- style="font-size:12pt" |style="background-color:#3366FF" height="15" | Tube ID: NP-BSPP_V5A_7P-Dec16 | align="center" align="center" | 181.37 | align="center" align="center" | 40 | align="center" align="center" | 221.37 | align="center" align="center" | 55.34 |} === Step 3. Gel Running & Size Selection Conditions=== *Run gel at 200V for 30mins *Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray. *Place tray on an orbital shaker for ~5 min. *Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. *Cut out the correct band (300-350 bp in this case), chop the band into small slices, pool two gel slices into one gel shearing assembly. *Change gloves and plastic wrap when size selecting a new gel. [[File:ZhangLab_2 2015-01-25 16hr 53min.jpg|400px]] [[File:ZhangLab_2 2015-01-25 16hr 51min.jpg|400px]] [[File:ZhangLab_2 2015-01-25 16hr 47min.jpg|400px]] [[File:ZhangLab_2 2015-01-25 18hr 28min.jpg|400px]] [[File:ZhangLab_2 2015-01-25 18hr 26min.jpg|400px]] [[File:ZhangLab_2 2015-01-25 18hr 24min.jpg|400px]] *Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. *Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *Spin down all tubes at 13,000rpm for 2min *In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube. ===Step 4. Precipitation=== '''Turn on centrifuge let it cool down to 4C''' *Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol. *Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA. *Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette. *Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette. *Let dry in hood ~ 10 min. *Add 25 uL of water per tube to resolve the DNA, leave at least 15 min. Store at 4C if needed, spin down before proceeding === Step 5. TBE Gel Size Verification === ===Step 6. Sequencing Prep=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | align="center" width="151" height="15" | | width="68" | nM | width="89" | To get 40nM | width="105" | Pooled Volume |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_6P Jan22 |style="background-color:#538DD5" align="center" | 34.87 |style="background-color:#538DD5" align="center" | 1.15 | n/a |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_7P Jan22 |style="background-color:#538DD5" align="center" | 19.85 |style="background-color:#538DD5" align="center" | 2.01 | n/a |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p1 Jan22 |style="background-color:#538DD5" align="center" | 26.08 |style="background-color:#538DD5" align="center" | 1.53 | n/a |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p2 Jan22 |style="background-color:#538DD5" align="center" | 12.23 |style="background-color:#538DD5" align="center" | 3.27 | n/a |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p3 Jan22 |style="background-color:#538DD5" align="center" | 34.25 |style="background-color:#538DD5" align="center" | 1.17 | n/a |- style="font-size:12pt" align="center" |style="background-color:#538DD5" height="15" | HL-BSPP V5A_PCP Jan22 |style="background-color:#538DD5" align="center" | 25.59 |style="background-color:#538DD5" align="center" | 1.56 | align="center" | 10.70 |- style="font-size:12pt" align="center" | height="15" | HL-Seqcap pCancerSetA Jan5 | align="center" | 85.88 | n/a | n/a |- style="font-size:12pt" align="center" | height="15" | HL-Seqcap pNormalSetA Jan5 | align="center" | 136.01 | n/a | n/a |}
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