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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] = Adaptor ligation of CoRE fragmented DNA (Eric's May 23, 2013 experiment) = * Got 24 samples from Eric for ligation using NEBNext adaptor. * MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May23, 2013: [[http://genome-tech.ucsd.edu/LabNotes/index.php/EricChu:LabNotesMDA/2013-5-23]] * 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 17-24 --> 24-17. * All procedures of adaptor ligation and amplification were the same as my experiment on May22, 2013 == 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 diluted a new batch of adaptor by diluting 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C after used. ''' 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;"|'''24x rxn mix''' |- | 2X KAPA Rapid Ligation Buffer||25.00||600.00 |- | KAPA Rapid T4 DNA Ligase||2.00||48.00 |- | H2O||15.33||367.92 |- | Total||42.33||1,015.92 |} * 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;"|'''25 rxn mix''' |- | adapter ligated DNA||20.00||0.00 |- | PCR_F (NEB, 10uM)||1.00||25.00 |- | NEBNext.IndX (NEB, 10uM)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||625.00 |- | H2O||3.00||75.00 |- | Total volume||50.00||725.00 |} * 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||#13||NEBNext Index1 |- | #2||NEBNext Index2||#14||NEBNext Index2 |- | #3||NEBNext Index3||#15||NEBNext Index3 |- | #4||NEBNext Index4||#16||NEBNext Index4 |- | #5||NEBNext Index5||#17||NEBNext Index5 |- | #6||NEBNext Index6||#18||NEBNext Index6 |- | #7||NEBNext Index7||#19||NEBNext Index7 |- | #8||NEBNext Index8||#20||NEBNext Index8 |- | #9||NEBNext Index9||#21||NEBNext Index9 |- | #10||NEBNext Index10||#22||NEBNext Index10 |- | #11||NEBNext Index11||#23||NEBNext Index11 |- | #12||NEBNext Index12||#24||NEBNext Index12 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C''' I stopped the reaction at '''11 cycles''' I plotted qPCR curves separately according to the gel loading order because I can't clearly observe amplification curve of each sample by plotting all 24 samples together. [[File:2013-05-24_Eric.qPCR_1-8.png| 325px]] [[File:2013-05-24_Eric.qPCR_9-16.png| 325px]] [[File:2013-05-24_Eric.qPCR_17-24.png| 325px]] * 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 2013-05-24 15hr 41min_1-8.jpg| 320px]] [[File:ZhangLab_2 2013-05-24 15hr 43min_9-16.jpg| 325px]] [[File:ZhangLab_2 2013-05-24 15hr 46min_17-24_e.jpg| 325px]] * All samples worked as expected since Eric did qPCR to verify the yield of MDA. * The average size of libraries looked smaller than the ones on May22 * This time, I reduced the number of cycle from 12 cycles to 11 cycles. Next time with the same amount of input, I may reduce to 9 or 10 cycles to reduce bias from amplification. <br> ''' PAGE quantification and sequencing library pooling ''' <br> * I did PAGE quantification on the gel images above (300-700bp range) and pooled 75ng each library index 1-12 together. ''' Pool 1: #1 - #12 ''' {| {{table}} border = 1 | 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||9.34||420.25||8.03 |- | #2||Index 2||8.48||381.66||8.84 |- | #3||Index 3||8.10||364.43||9.26 |- | #4||Index 4||9.05||407.25||8.29 |- | #5||Index 5||8.09||363.98||9.27 |- | #6||Index 6||8.59||386.41||8.73 |- | #7||Index 7||10.35||465.54||7.25 |- | #8||Index 8||8.22||370.02||9.12 |- | #9||Index 9||9.30||418.65||8.06 |- | #10||Index 10||8.51||382.75||8.82 |- | #11||Index 11||10.79||485.45||6.95 |- | #12||Index 12||10.12||455.39||7.41 |} ''' Pool 2: #13 - #24 ''' {| {{table}} border = 1 | 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)''' |- | #13||Index 1||10.65||479.03||7.05 |- | #14||Index 2||13.01||585.60||5.76 |- | #15||Index 3||11.26||506.66||6.66 |- | #16||Index 4||10.77||484.48||6.97 |- | #17||Index 5||7.14||321.12||10.51 |- | #18||Index 6||10.48||471.75||7.15 |- | #19||Index 7||10.66||479.57||7.04 |- | #20||Index 8||8.76||394.04||8.57 |- | #21||Index 9||12.23||550.38||6.13 |- | #22||Index 10||11.96||538.07||6.27 |- | #23||Index 11||8.76||394.36||8.56 |- | #24||Index 12||11.12||500.41||6.74 |}<br> ''' 2013_05_25 '''<br> ''' PAGE size-selection ''' * Each pool has ~900ng of 300-700bp fragments * Each pool purified in 1 of 6% 5-well TBE gel [[File:ZhangLab_2 2013-05-25 18hr 06min_ss-pool1.jpg| 300px]] [[File:ZhangLab_2 2013-05-25 18hr 13min_ss-pool2.jpg| 300px]] I loaded too much 100bp DNA ladder (500ng, 50ng/ul). The bands are too close, so difficult to locate 700bp band. Next time may load 250ng and run a little longer for a better resolution. * Resuspend each pool with total 40ul H2O <br> ''' PAGE verification of sequencing libraries ''' * Loaded 1.5ul of each library pool in 6% TBE gel [[File:ZhangLab_2 2013-05-27 13hr 04min_EC-PGP1-PAGEverify.jpg| 150px]] P1 = EC-PGP1-Dev7-1-12-May23-2013 P2 = EC-PGP1-Dev7-13-24-May23-2013 *** This time, I cut the gel lower than expected 700bp bands (~600bp)!! ''' Library ID, Pool1: EC-PGP1-Dev7-1-12-May23-2013 : ''' conc. by Qubit dsDNA HS (used 2ul for assay) = 3.51 ng/ul or '''10.79 nM''' <br> ''' Library ID, Pool2: EC-PGP1-Dev7-13-24-May23-2013 :''' conc. by Qubit dsDNA HS (used 2ul for assay) = 3.56 ng/ul or '''10.95 nM''' <br> * MW of 500bp (average of 300-700bp) = 500bp * 650Da/bp + 159Da ~ 325,159Da or g/mole * Alan did qPCR to quantify the concentration of the libraries again '''GAII run: HL153 (SE)''' * EC-PGP1-Dev7-1-12-May23-2013: Lane 3 * EC-PGP1-Dev7-13-24-May23-2013: Lane 4
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