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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/2013-1-11 = Skin Microbiom Project = = Library construction = For details of each step, epMotion log file will be added later. Protocol's name: XXX * Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer and keep the bead in the well. (Note: since the beads will be reused during library prep, I didn't dilute the beads). * Note: Lost sample in well A1 (well broken) * Dr. Zhang suggested to prepare 7 libraries, but keep 3 of them (F1-H1) to be used later and do PCR on 4 samples (B1-E1) * Estimate concentration by PAGE quantification (to use this amount as the average amount in the future experiment by assume that each sample has equal amount of DNA. [[File:ZhangLab_2 2013-01-15 12hr 28min_purified_shearedDNA.jpg| 400px]] {| {{table}} | align="center" style="background:#f0f0f0;"|'''WellID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 50ul''' |- | B1||0.26||13.21 |- | C1||1.60||79.87 |- | D1||3.92||196.11 |- | E1||2.57||128.49 |- | F1||4.67||233.58 |- | G1||1.56||78.03 |- | H1||1.80||90.23 |} * From the concentration above, the total amount of amlpicons is not as high as 1ug, but in the range ~20-230ng. Taking the average of the total amount of all 7 samples, ~100ng total. ==DISCUSSION == (about total DNA amount and problem of small volume DNA transfer to Covaris shearing plate)<br> XXXX == 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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8x rxn mix''' |- | 10X End repai buffer||7.00||56.00 |- | KAPA End repair enzyme||5.00||40.00 |- | H2O||8.00||64.00 |- | Total||20.00||160.00 |} * Aliquot to column #9: 22ul == 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;"|'''8x rxn mix''' |- | KAPA 10X A-tailing buffer||3.00||24.00 |- | KAPA A-tailing enzyme||3.00||24.00 |- | H2O||24.00||192.00 |- | Total||30.00||240.00 |} * Aliquot to column #10: 32ul == Adapter ligation == * Adapter ligation mix: Total 40ul/reaction -> 10ul is adapters * 20C for 15min<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8x rxn mix''' |- | KAPA 5 Ligation buffer||10.00||80.00 |- | KAPA DNA ligase||4.00||32.00 |- | H2O||26.00||208.00 |- | Total||40.00||320.00 |} * Aliquot to column #11: 43ul == Adapter preparation == Assume DNA amount ~100ng, size 400bp MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243,118 g/mole For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required. 20uM adapters (ul) required = 0.0082nmoles/(20xE3 nmoles/L * 1E-06L/ul) = 0.0082nmoles/(20E-3 nmoles/ul) = 0.411ul I will prepare total volume of 20uM annealed adapters 10ul {| {{table}} | Stoffel buffer||1.00 |- | PE_t_adapter||2.00 |- | PE_b_adapter||2.00 |- | H2O||5.00 |} * 94C 2min -> 0.2C/sec to 20C -> 4C hold * Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> anneal adapterr 4.1ul: 1x Stoffelul 95.9 {| {{table}} | 20uM annealed adapter||4.10 |- | 10x Stoffel buffer||10.00 |- | H2O||85.90 |} * Aliquot to column #12: 13ul * After finish adapter ligation, adapter ligated DNA was resuspended in 15ul EB buffer == Amplification == * '''TEST''' if amplification works (in small volume 25ul add 1.25ul of adapter ligated template) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''6x rxn mix''' |- | adapter ligated DNA||1.25||0.00 |- | PCR_F(10uM)||0.50||3.00 |- | PCR_R (10uM)||0.50||3.00 |- | SYBR Green 50x||0.10||0.60 |- | H2O||10.15||60.90 |- | Phusion HF, 2xMM||12.50||75.00 |- | Total volume||25.00||150.00 |} * Aliquot 23.75ul, add 1.25 of adapter ligated DNA '''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C [[File:qPCR_20130115_TESTpcr.png.png| 500px]] * PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification) [[File:ZhangLab_2 2013-01-15 21hr 12min_Test_PCR.jpg| 300px]] * Since PCR worked well, I amplified all adapter ligated DNA (B1-E1, 3 replicates, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13.5rxn mix''' |- | adapter ligated DNA||5.00||0.00 |- | PCR_F(10uM)||2.00||27.00 |- | PCR_R.N2IndX(10uM)[X=1-4]||2.00||0.00 |- | SYBR Green 50x||0.80||10.80 |- | H2O||40.20||542.70 |- | Phusion HF, 2xMM||50.00||675.00 |- | Total volume||100.00||1350.00 |} * Aliquot 93ul, add 5 of adapter ligated DNA and 2ul of PCR_R.N2Indx '''Program''' 98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C [[File:qPCR_20130115_3-replictes_B1-E1.png.png| 500px]] * Note: I used different types of plates (TEST PCR used the real time PCR tube (Eppendort WW), the 3 replicates used the Eppendorf plate for epMotion by set up the back ground as the clear plate) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position in<br>AC-718-384-well''' | align="center" style="background:#f0f0f0;"|'''Position in<br> AC-718-96-well_plate1''' | align="center" style="background:#f0f0f0;"|'''Individual library ID''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | J03||B1||718_plate1_B1||PCR_R.N2Indx1 |- | L02||C1||718_plate1_C1||PCR_R.N2Indx2 |- | M05||D1||718_plate1_D1||PCR_R.N2Indx3 |- | N03||E1||718_plate1_E1||PCR_R.N2Indx4 |} * Pooled 80ul of amplified amplicons from the three replicates (total 240ul) and bead purified (diluted Ampure beads 3X with 20%PEG) with epMotion. * Eluted with EB buffer total volme 75ul * Dr. Zhang suggested to pooled 50% of each library and perform PAGE size-selection (may not equal molar ratio). [[File:ZhangLab_2 2013-01-17 13hr 37min_718_plate1_B1-E1-PAGE-SS.jpg| 500px]] * I cut the band at the position between 400-600bp, resuspend sequencing library with H2O total volume 30ul. * Alan will do qRCR to quantify the library concentration * Verify the library by PAGE analysis [[File:ZhangLab_2 2013-01-17 16hr 07min_SkinMB_718_p1_N2_B1-E1_Jan15-e.jpg| 300px]] * '''Library ID: JG-SkinMB_718_p1_N2_B1-E1_Jan15.2013''' * I performed PAGE quantification on individual library (B1-E1) in the range 400-600bp for further experiment (DSN) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Position in <br>AC-718-384-well''' | align="center" style="background:#f0f0f0;"|'''Position in <br>AC-718-96-well_plate1''' | align="center" style="background:#f0f0f0;"|'''Individual library ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 35ul (ng)''' |- | J03||B1||718_plate1_B1||0.88||30.86 |- | L02||C1||718_plate1_C1||2.59||90.55 |- | M05||D1||718_plate1_D1||3.12||109.08 |- | N03||E1||718_plate1_E1||2.74||95.82 |} * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-31
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