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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-16 == Low input (1ng) library preparation using Kapa kit (updated) == === 2013_04_23 === * Even Jeff will process his MDA products using CGI protocol, we still want to try the standard protocol following Rui's condition she used for her experiment. * Things have been adjusted ** DNA shearing: I included the 200bp fragment to compare if the size effects adapter ligation efficiency (Rui: 200bp fragment) ** Prepared the same concentration of adapter (30uM) and used the same concentration for ligation ** Amplification: *** Annealing temperature: I previously used 65C for annealing. I reduced annealing temperature to 62C (Rui: 60C with the same pair of primers). *** Reduced volume from 100ul to 50ul *** Slightly changes were noted during each step * Sample list: I still included higher amount of DNA (2.5 & 5ng) to compare the improvement by adjusting the conditions. ** NTC, 1ng_200bp, 2.5ng_200bp, 5ng_200bp, 1ng_400bp, 2.5ng_400bp, 5ng_400bp == 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 |} {| 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;"|'''7x rxn mix''' |- | 10X End repai buffer||7.00||49.00 |- | KAPA End repair enzyme||3.50||24.50 |- | H2O||9.50||66.50 |- | Total||20.00||140.00 |} * 20C for 30min (no shaking) * Bead purification: 112ul AMPure beads, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min * Add 20ul of H2O to resuspend the beads first to make sure that all DNAs were completely dissolved (keep the beads) before adding A-tailing reaction mix == A-tailing == * A-tailing reaction mix: Total 50ul/reaction (increase from 30ul to 50ul) * DNAs were dissolved with 20ul H2O in the previous step (the beads were carried over till the last step) {| {{table}} | align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''7x rxn mix''' |- | KAPA 10X A-tailing buffer||5.00||35.00 |- | KAPA A-tailing enzyme||3.00||21.00 |- | H2O||22.00||154.00 |- | Total||30.00||210.00 |} * Add 30ul of A-tailing reaction mix to 20ul dissolved DNA * 30C for 30min (no shaking) * Bead purification: 90ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min * Add 20ul of H2O to resuspend the beads as described in A-tailing (keep the beads) before adding ligation reaction mix and adapters == Adapter ligation == * Adapter ligation mix (Total 40ul) : Ligation reaction mix 39.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer) * DNAs were dissolved with 20ul H2O in the previous step {| 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;"|'''7x rxn mix''' |- | KAPA 5X Ligation buffer||8.00||56.00 |- | KAPA DNA ligase||4.00||28.00 |- | H2O||7.20||50.40 |- | Total||19.20||134.40 |} * Add 19.2ul of Ligation reaction mix to 20ul dissolved DNA * Add 0.8ul of 3uM adapters * 20C for 15min (no shaking) * Bead purification: 50ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min * Eluted with 15ul H2O (used all for PCR) == Amplification == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8.5 rxn mix''' |- | adapter ligated DNA||15.00||0.00 |- | PCR_F(10uM)||1.00||8.50 |- | PCR_R.N2IndX(10uM)||1.00||0.00 |- | H2O||8.00||68.00 |- | 2X KAPA SYBR MM||25.00||212.50 |- | Total volume||50.00||425.00 |} * Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | 1ng_200bp||PCR_R.N2Indx1 |- | 2.5ng_200bp||PCR_R.N2Indx2 |- | 5ng_200bp||PCR_R.N2Indx3 |- | 1ng_400bp||PCR_R.N2Indx4 |- | 2.5ng_400bp||PCR_R.N2Indx5 |- | 5ng_400bp||PCR_R.N2Indx6 |- | NTC1 with adapters||PCR_R.N2Indx7 |- | NTC2 no adapters||PCR_R.N2Indx8 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 62C - 30s, 72C - 30s)x20, 72C - 2min, hold at 15C * I set up total 20 cycles, but monitored to stop at any number of cycle before reaching a plateau * Stop at '''13 cycles''' '''qPCR curve''' [[File:20130424_qPCR_LowInpu.png |550px]] * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) [[File:20130424_qPCR_LowInput.png| 330px]] * From the qPCR curve and gel image, the background from adapters was very high comparing to the signal of expected bands. The qPCR curves between NTC and experiment were very close for very low input. Verification by PAGE telling more information * 2.5ng and 5ng clearly showed amplification. The background should be clean after bead purification. Still did not see amplification for 1ng sample either 200bp or 400bp *These conditions show more improvement comparing to previous experiment with 20 cycles. * No significant different between 200bp and 400bp DNA fragments. Seem like 400bp show slightly stronger signal. * The signal was lower than Rui with 15 cycles amplification. Rui's result showed stronger signal than background. I might optimize annealing temperature by using two step PCRs to minimize the background form adapters. * The amount of unligated adapters after ligation reaction interfere qPCR curve. * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-25
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