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= Adaptor ligation of CoRE fragmented DNA (Eric's May 21, 2013 experiment) = * Got 16 samples from Eric for ligation using NEBNext adaptor. (Samples were already in 0.2 ml PCR Tube Strips, low-Profile tube (Biorad)) * MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May21, 2013: [[http://genome-tech.ucsd.edu/LabNotes/index.php/EricChu:LabNotesMDA/2013-5-21]] * Note that the order of samples in 8-striptube ordered by Eric and me was in the opposite way within the striptube. So number 1-8 by Eric will be flipped to 8-1 by me and also for 19-16 --> 16-9. == Adaptor ligation == ''' Reaction set up''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | Fragmented DNA||6.00 |- | 1.5uM NEBNext adaptor||1.67 |- | Ligation reaction mix||42.33 |- | Total||50.00 |} ''' NEBNext adaptor preparation ''' * I used 1.5uM adaptor prepared in the previous experiment * I diluted 15uM adaptor to 1.5uM (mix 10ul of 15uM NEBNext adaptor with 90ul H2O) and aliquoted 12ul into 8-striptubes and saved at -20C * The concentration of adaptor for ultra-low input DNA (<=10ng) in 50ul reaction is 50nM ''' Ligation reaction mix ''' {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''16x rxn mix''' |- | 2X KAPA Rapid Ligation Buffer||25.00||400.00 |- | KAPA Rapid T4 DNA Ligase||2.00||32.00 |- | H2O||15.33||245.28 |- | Total||42.33||677.28 |} * Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA * Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin. * 25C for 10min * Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min ** For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less '''Bead purification''' * 50ul AmPure beads mix by pipetting 10 times * Wait 10min * Sit on magnetic stand 5min * Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt) ** I wash with 160ul of 80% EtOH since I used low profile tube. * Dry the beads for 3 min * Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR) == Amplification == {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''16.5 rxn mix''' |- | adapter ligated DNA||20.00||0.00 |- | PCR_F (NEB, 10uM)||1.00||16.50 |- | NEBNext.IndX (NEB, 10uM)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||412.50 |- | H2O||3.00||49.50 |- | Total volume||50.00||478.50 |} * Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | #1||NEBNext Index1||#9||NEBNext Index1 |- | #2||NEBNext Index2||#10||NEBNext Index2 |- | #3||NEBNext Index3||#11||NEBNext Index3 |- | #4||NEBNext Index4||#12||NEBNext Index4 |- | #5||NEBNext Index5||#13||NEBNext Index5 |- | #6||NEBNext Index6||#14||NEBNext Index6 |- | #7||NEBNext Index7||#15||NEBNext Index7 |- | #8||NEBNext Index8||#16||NEBNext Index8 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C''' I stopped the reaction at '''12 cycles''' [[File:20130522_qPCR_CoRE.png| 650px]] * Eric should provide the sample info. * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) [[File:ZhangLab_2 2013-05-22 13hr 33min_gel1_1-8.jpg| 420px]] [[File:ZhangLab_2 2013-05-22 13hr 35min_gel2_9-18.jpg| 420px]] * Work for all samples, but the library size was a little high (average ~800bp) than the library got from Jeff's experiment in April30, 2013. ''' 2013_05_23 ''' * Dr. Zhang suggested to pool Index1-8 (two pools because of the index duplicates) together, size-select '''300-700bp''' fragments, and do '''5 million SE36bp reads per pool''' ''' PAGE quantification and sequencing library pooling ''' <br> * I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library. * Note, I din't do bead purification before PAGE size-selection. However, if libraries needed to be saved for future experiment, I would do bead purify. ''' Pool1: #1 - #8 ''' {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 45ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 75ng (ul)''' |- | #1||Index 1||8.51||383.13||8.81 |- | #2||Index 2||9.05||407.32||8.29 |- | #3||Index 3||3.86||173.74||19.43 |- | #4||Index 4||5.12||230.32||14.65 |- | #5||Index 5||6.96||313.37||10.77 |- | #6||Index 6||7.52||338.38||9.97 |- | #7||Index 7||9.05||407.12||8.29 |- | #8||Index 8||6.72||302.42||11.16 |} ''' Pool2: #9 - #16 ''' {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 45ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 75ng (ul)''' |- | #9||Index 1||5.80||260.82||12.94 |- | #10||Index 2||5.93||266.90||12.65 |- | #11||Index 3||8.04||361.67||9.33 |- | #12||Index 4||7.12||320.47||10.53 |- | #13||Index 5||4.42||199.01||16.96 |- | #14||Index 6||8.48||381.72||8.84 |- | #15||Index 7||6.84||307.85||10.96 |- | #16||Index 8||7.76||349.40||9.66 |} ''' PAGE size-selection ''' * Each pool has ~600ng of 300-700bp fragments * Each pool purified in 1 of 6% 5-well TBE gel [[File:ZhangLab_2 2013-05-23 13hr 47min_SS-pool1_e.jpg| 300px]] [[File:ZhangLab_2 2013-05-23 13hr 50min_aa-pool2.jpg| 300px]] * Resuspend each pool with total 30ul H2O ''' PAGE verification of sequencing libraries ''' * Loaded 1.5ul of each library pool in 6% TBE gel [[File:ZhangLab_2 2013-05-23 19hr 13min_EC-MEF-PAGE.jpg| 150px]] P1 = EC-MEF-Dev7-1-8-May21-2013 P2 = EC-MEF-Dev7-9-16-May21-2013 *** It seems like I cut the gel too close to 800bp especially library pool2 *** To cut the size precisely, may need to change DNA ladder like 100bp DNA ladder to locate the 300bp and 700bp fragments ''' Library ID, Pool1: EC-MEF-Dev7-1-8-May21-2013 ''': conc. by Qubit dsDNA HS (used 2ul for assay) = 3.20 ng/ul or '''9.84nM''' <br> ''' Library ID, Pool2: EC-MEF-Dev7-9-16-May21-2013 ''': conc. by Qubit dsDNA HS (used 2ul for assay) = 4.60 ng/ul or '''14.15nM'''<br> * MW of 500bp (average of 300-700bp) = 500bp * 650Da/bp + 159Da ~ 325159Da or g/mole * Alan did qPCR to quantify the concentration of the libraries again ''' GAII run: HL153 (SE)''' * EC-MEF-Dev7-1-8-May21-2013: Lane 1 * EC-MEF-Dev7-9-16-May21-2013: Lane 2
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