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= Library construction (72 samples) = * After DNA shearing on 96-well microtubes, I did bead purification and resuspend DNA in 50ul. ** There were some problem during transfer samples from micro-tubes to 96-well plate. The aluminum foil I used to seal the plate had a very bad quality, since it left the sticky part cover the well after I unsealed it. This block the pipette tip to transfer the liquid. I need to manually make the whole with clean pipette tip to clear the well before transfering sample with the robot. Another problem is the robot didn't completely transfer the liquid to the well (may be I need to adjust the dispensing speed). It have the droplet at the tip and might drop and get contaminate to other wells (I observed this about 3-4 samples) * I randomly pick 10 samples to run the gel check (I used little amount of beads (saw the bead left in the well very little) but comparable to the previous time, so I'm kind of worried about if I will lose most of the DNA). Next time I may use undiluted beads instead since it will be re-used in the following steps of library prep. [[File:ZhangLab_2 2013-02-07 12hr 45min_check_shearedDNA_aftersheared_beadpure.jpg| 450px]] * From the gem image, I got the intensity of DNA in all 10 samples, and it's pretty close to the rehearsal. == End repair == {| {{table}} | width ="150pt" align="center" style="background:#f0f0f0;"|'''Total reaction''' | align="center" style="background:#f0f0f0;"|'''70.00''' | align="center" style="background:#f0f0f0;"|'''ul''' |- | DNA ||50.00||ul |- | End repair reaction mix ||20.00||ul |} * 20C for 30min {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''75x rxn mix''' |- | 10X End repai buffer||7.00||525.00 |- | KAPA End repair enzyme||5.00||375.00 |- | H2O||8.00||600.00 |- | Total||20.00||1500.00 |} * Aliquot to column #1: 187ul == A-tailing == * A-tailing reaction mix: Total 30ul/reaction * 30C for 30min {| {{table}} | align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''75x rxn mix''' |- | KAPA 10X A-tailing buffer||3.00||225.00 |- | KAPA A-tailing enzyme||3.00||225.00 |- | H2O||24.00||1800.00 |- | Total||30.00||2250.00 |} * Aliquot to column #2 (for 40rxn): 155ul, column #3 (for 32 rxn): 124ul == Adapter ligation == * Adapter ligation mix (Total 50ul) : Ligation reaction mix 40ul/reaction & 10ul of adapters * 20C for 15min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''75x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||750.00 |- | KAPA DNA ligase||3.00||225.00 |- | H2O||27.00||2025.00 |- | Total||40.00||3000.00 |} * Aliquot to column #4 (for 40rxn): 207ul, column #5 (for 32 rxn): 165ul == Adapter preparation == Assume DNA amount ~100ng, size 400bp MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243118 g/mole For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required. Adapters (ul) = 0.0082nmoles/ (20xE3 nmoles/L * 1E-6L/ul) = 0.0082nmoles/ (20E-3 nmoles/ul) = 0.411ul * I will prepare total volume of 20uM annealed adapters 40ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''10ul''' | align="center" style="background:#f0f0f0;"|'''40ul''' |- | Stoffel buffer||1.00||4.00 |- | PE_t_adapter||2.00||8.00 |- | PE_b_adapter||2.00||8.00 |- | H2O||5.00||20.00 |} * 94C 2min -> 0.2C/sec to 20C -> 4C hold * Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> I will prepare diluted adapters for ~90rxn) {| {{table}} | 20uM annealed adapter||4.10 (for 10 rxn||36.90 (for ~90 rxn) |- | 10x Stoffel buffer||10.00||90.00 |- | H2O||85.90||773.10 |- | Total||100.00||900.00 |} * Aliquot to column #6 = 112ul == Amplification == * '''TEST''' if amplification works (in small volume 25ul add 3ul of adapter ligated template) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''20.00''' |- | adapter ligated DNA||1.25||0.00 |- | PCR_F(10uM)||0.50||10.00 |- | PCR_R.N2IndX(10uM)||0.50||10.00 |- | H2O||10.25||205.00 |- | 2X KAPA SYBR MM||12.50||250.00 |- | Total volume||25.00||500.00 |} ''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x10, 72C - 2min, hold at 15C * PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification) [[File:ZhangLab_2 2013-02-08 16hr 24min_TEST-PCR_A3-H3_A4-H4_2.jpg| 450px]] * I prepared PCR mix using KAPA SYBR fast MM (amplified 2 plates with identical conditions) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''80 rxn mix''' |- | adapter ligated DNA||16.00||0.00 |- | PCR_F(100uM)||0.20||16.00 |- | PCR_R.N2IndX(5uM)||4.00||0.00 |- | H2O||29.80||2,384.00 |- | 2X KAPA SYBR MM||50.00||4,000.00 |- | Total volume||100.00||8,000.00 |} Program 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x10, 72C - 2min, hold at 15C == Sequencing library concentration by Nanodrop == * Note: To combine equal molecular ratio of sequencing libraries, I measured concentration of each library by Nanodrop. The corresponding concentration may not be very accurate as PQ (at specific 300-600bp range) and the concentration may be overestimated. However, this should be good enough. For some libraries which have very low concentration (<5.5ng/ul), I add maximum volume 35ul to the library pool to save half of them for DSN nomalization. {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''Yields in 70ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)<br>pooled in the seqlib''' | align="center" style="background:#f0f0f0;"|'''Approximated volume left (ul)''' |- | a3||10.51||735.70||19.03||200.00||50.97 |- | b3||12.93||905.10||15.47||200.00||54.53 |- | c3||7.86||550.20||25.45||200.00||44.55 |- | d3||6.56||459.20||30.49||200.00||39.51 |- | f3||12.22||855.40||16.37||200.00||53.63 |- | f3||12.60||882.00||15.87||200.00||54.13 |- | g3||5.48||383.60||35.00||191.80||35.00 |- | h3||8.37||585.90||23.89||200.00||46.11 |- | a4||10.34||723.80||19.34||200.00||50.66 |- | b4||16.43||1150.10||12.17||200.00||57.83 |- | c4||40.51||2835.70||4.94||200.00||65.06 |- | d4||30.31||2121.70||6.60||200.00||63.40 |- | e4||20.63||1444.10||9.69||200.00||60.31 |- | f4||22.15||1550.50||9.03||200.00||60.97 |- | g4||15.48||1083.60||12.92||200.00||57.08 |- | h4||13.09||916.30||15.28||200.00||54.72 |- | a5||13.60||952.00||14.71||200.00||55.29 |- | b5||29.56||2069.20||6.77||200.00||63.23 |- | c5||15.63||1094.10||12.80||200.00||57.20 |- | d5||30.58||2140.60||6.54||200.00||63.46 |- | e5||29.43||2060.10||6.80||200.00||63.20 |- | f5||16.55||1158.50||12.08||200.00||57.92 |- | g5||11.05||773.50||18.10||200.00||51.90 |- | h5||12.59||881.30||15.89||200.00||54.11 |- | a6||12.06||844.20||16.58||200.00||53.42 |- | b6||18.89||1322.30||10.59||200.00||59.41 |- | c6||12.57||879.90||15.91||200.00||54.09 |- | d6||13.98||978.60||14.31||200.00||55.69 |- | e6||18.46||1292.20||10.83||200.00||59.17 |- | f6||18.98||1328.60||10.54||200.00||59.46 |- | g6||12.28||859.60||16.29||200.00||53.71 |- | h6||4.43||310.10||35.00||155.05||35.00 |- | a7||9.23||646.10||21.67||200.00||48.33 |- | b7||20.87||1460.90||9.58||200.00||60.42 |- | c7||19.38||1356.60||10.32||200.00||59.68 |- | d7||33.00||2310.00||6.06||200.00||63.94 |- | e7||16.55||1158.50||12.08||200.00||57.92 |- | f7||16.68||1167.60||11.99||200.00||58.01 |- | g7||13.04||912.80||15.34||200.00||54.66 |- | h7||9.28||649.60||21.55||200.00||48.45 |- | a8||5.89||412.30||33.96||200.00||36.04 |- | b8||9.28||649.60||21.55||200.00||48.45 |- | c8||10.88||761.60||18.38||200.00||51.62 |- | d8||10.91||763.70||18.33||200.00||51.67 |- | e8||10.18||712.60||19.65||200.00||50.35 |- | f8||10.30||721.00||19.42||200.00||50.58 |- | g8||3.65||255.50||35.00||127.75||35.00 |- | h8||4.04||282.80||35.00||141.40||35.00 |- | a9||5.71||399.70||35.03||200.00||34.97 |- | b9||7.70||539.00||25.97||200.00||44.03 |- | c9||13.63||954.10||14.67||200.00||55.33 |- | d9||15.94||1115.80||12.55||200.00||57.45 |- | e9||10.19||713.30||19.63||200.00||50.37 |- | f9||14.79||1035.30||13.52||200.00||56.48 |- | g9||14.80||1036.00||13.51||200.00||56.49 |- | h9||10.99||769.30||18.20||200.00||51.80 |- | a10||4.78||334.60||35.00||167.30||35.00 |- | b10||10.83||758.10||18.47||200.00||51.53 |- | c10||7.88||551.60||25.38||200.00||44.62 |- | d10||13.33||933.10||15.00||200.00||55.00 |- | e10||8.53||597.10||23.45||200.00||46.55 |- | f10||10.81||756.70||18.50||200.00||51.50 |- | g10||10.17||711.90||19.67||200.00||50.33 |- | h10||6.92||484.40||28.90||200.00||41.10 |- | a11||6.90||483.00||28.99||200.00||41.01 |- | b11||9.62||673.40||20.79||200.00||49.21 |- | c11||8.67||606.90||23.07||200.00||46.93 |- | d11||12.25||857.50||16.33||200.00||53.67 |- | e11||11.38||796.60||17.57||200.00||52.43 |- | f11||11.91||833.70||16.79||200.00||53.21 |- | g11||12.44||870.80||16.08||200.00||53.92 |- | h11||9.89||692.30||20.22||200.00||49.78 |} * Performed PAGE size-selection (200ng each from 72 libraries, in 4 of 5-well gels) [[File:ZhangLab_2 2013-02-12 12hr 21min_PAGE-ss1.jpg| 450px]] [[File:ZhangLab_2 2013-02-12 12hr 24min_PAGE-ss2.jpg| 480px]] * Resuspend ~70ul with H20 * Verify the size in 6% TBE gel [[File:ZhangLab_2 2013-02-12 15hr 59min.jpg| 240px]] * Quantify concemtration by Qubit dsDNA HS assay ** Average conc. of = 19.73ng/ul ** Conc. based on MW (size ~400bp) 243,228g/mol = 81.1nM * '''Library ID: NP-SkinMB_N2_Ind17-88_Feb07.2013'''. The libraires were sequenced in HL144 run (lane1), HL145 (lane ) * For sample tracking, please refer to the table on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-31 * The plate labeled: ''' SkinMB.Ind17-88.Feb07,2013''' {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Position in 96-well''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Position in 96-well''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | A3||PCR_R.N2Indx17||E7||PCR_R.N2Indx53 |- | B3||PCR_R.N2Indx18||F7||PCR_R.N2Indx54 |- | C3||PCR_R.N2Indx19||G7||PCR_R.N2Indx55 |- | D3||PCR_R.N2Indx20||H7||PCR_R.N2Indx56 |- | E3||PCR_R.N2Indx21||A8||PCR_R.N2Indx57 |- | F3||PCR_R.N2Indx22||B8||PCR_R.N2Indx58 |- | G3||PCR_R.N2Indx23||C8||PCR_R.N2Indx59 |- | H3||PCR_R.N2Indx24||D8||PCR_R.N2Indx60 |- | A4||PCR_R.N2Indx25||E8||PCR_R.N2Indx61 |- | B4||PCR_R.N2Indx26||F8||PCR_R.N2Indx62 |- | C4||PCR_R.N2Indx27||G8||PCR_R.N2Indx63 |- | D4||PCR_R.N2Indx28||H8||PCR_R.N2Indx64 |- | E4||PCR_R.N2Indx29||A9||PCR_R.N2Indx65 |- | F4||PCR_R.N2Indx30||B9||PCR_R.N2Indx66 |- | G4||PCR_R.N2Indx31||C9||PCR_R.N2Indx67 |- | H4||PCR_R.N2Indx32||D9||PCR_R.N2Indx68 |- | A5||PCR_R.N2Indx33||E9||PCR_R.N2Indx69 |- | B5||PCR_R.N2Indx34||F9||PCR_R.N2Indx70 |- | C5||PCR_R.N2Indx35||G9||PCR_R.N2Indx71 |- | D5||PCR_R.N2Indx36||H9||PCR_R.N2Indx72 |- | E5||PCR_R.N2Indx37||A10||PCR_R.N2Indx73 |- | F5||PCR_R.N2Indx38||B10||PCR_R.N2Indx74 |- | G5||PCR_R.N2Indx39||C10||PCR_R.N2Indx75 |- | H5||PCR_R.N2Indx40||D10||PCR_R.N2Indx76 |- | A6||PCR_R.N2Indx41||E10||PCR_R.N2Indx77 |- | B6||PCR_R.N2Indx42||F10||PCR_R.N2Indx78 |- | C6||PCR_R.N2Indx43||G10||PCR_R.N2Indx79 |- | D6||PCR_R.N2Indx44||H10||PCR_R.N2Indx80 |- | E6||PCR_R.N2Indx45||A11||PCR_R.N2Indx81 |- | F6||PCR_R.N2Indx46||B11||PCR_R.N2Indx82 |- | G6||PCR_R.N2Indx47||C11||PCR_R.N2Indx83 |- | H6||PCR_R.N2Indx48||D11||PCR_R.N2Indx84 |- | A7||PCR_R.N2Indx49||E11||PCR_R.N2Indx85 |- | B7||PCR_R.N2Indx50||F11||PCR_R.N2Indx86 |- | C7||PCR_R.N2Indx51||G11||PCR_R.N2Indx87 |- | D7||PCR_R.N2Indx52||H11||PCR_R.N2Indx88 |} * Continued on : [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-20]], pooled the top 20 libraries for HiSeq sequencing
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