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= Expansion PCR of BSPP from CustomArrays 90K_ologos_04Aug2014 (8-20-14) = * [[noi:DMR220k_LabNotes| '''Back to calendar''']] * 2014-08-13: Received MOMOD V5 Oligo pool from CustomAraay === Oligo info. === * Name: 90K_ologos_04Aug2014 (8-20-14) * Conc. 153.49ng/ul, * Volume 80ul in TE buffer. Total amount 12.28ug * Info from Dr. Zhang's note Description # oligos Length Amp. Primers Adaptor Removal Set A: BSPP LMS cluster probes 73,574 148nt V6 USER/DpnII Set B: Selector LMS cluster 12,994 148nt V4 USER/DpnII Set C: Selector chr21 unique 2,539 148nt eMIP_CA_F/R Nt.AlwI/Nb.BrsDI Total: 89,107 * I will be working on 73K probe set * Length 148 mer (MW = 148 * 330Da/bp = 48,840 Da or g/mole * Conc. 20.53ng/ul = 3.1427uM or 3142.7nM (153.49ng/ul/ 48,840g/mole) <br> == Expansion PCR (Quick test, round1) == * I did a quick test of expansion PCR on the three subset (20ul reaction). * I diluted 1st amplicon of previous probe set amplified by each primer set (by V4, V6, or eMIP_CA primers) into 2nM and will be as positive control for amplification. ** V4 set: I used MONOD GP1V4 as template. Mix 10ul of 20nM 1st round amplicon MONOD GP1V4 with 90ul H2O ** V6 set: I used MONOD V3 as template. Prepare 2nM template in the same way as V4 set. ** eMIP_CA: I used oligo from LC sciences used in Nature methods 2012. Mix 20ul of 10nM 1st round amplicon 4K LC Sciences with 80ul H2O 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 18-> 72C 3min -> 15C hold<br> [[File:2014-08-21_test_ExpPCR-90k_18x-r1.PNG|600px]] * Loaded 3ul of positive control and 5ul of amplicons from each subset in 6% TBE gel [[File:ZhangLab_2 2014-08-21 15hr 36min_90K-testExpPCR_r1.jpg|400px]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:100px;background:#f0f0f0;"|'''# oligos''' | align="center" style="width:100px;background:#f0f0f0;"|'''% in the pool''' |- | Set A||73,574||82.57 |- | Set B||12,994||14.58 |- | Set C||2,539||2.85 |} * Based on qPCR curve and gel image of the largest subset, subset A (82.57%), it looked like the amplification efficiency was not as high as previous batches of oligos from CustomArray. For optimization of expansion PCR in Blueprint project and other batches of oligos from CustomArray, qPCR curve started to shoot up ~6-7 cycles for 100nM oligo template and the band intensity was stronger by loading only 2.5ul of amplicons. * The smaller subset (set B and set C) had very faint intensity, so Rui wanted to know if increasing template amount would increase the intensity of the 150bp amplicons without inhibiting PCR reaction. I then tried a quick PCR again by increasing template concentration of set B to 150nM and reduce PCR cycle to 16 and 17 cycles. For subset C, I increase template concentration to 150nM and 200nM and ran PCR for 18 cycles since it takes only 2.85% of the pools. For subset A, I reduced PCR cycle number to 15 and 16 cycles, but used the same concentration of 100nM. ---- === Result of expansion PCR in Blueprint project (for comparison) === [[File:20130530_BSPPBlueprint_TESTExpensionPCR.png| 550px]] [[File:ZhangLab_2 2013-05-30 15hr 54min_TESTExpansionPCR.jpg| 200px]] [[File:20130530_BSPPBlueprint_ExpensionPCR.png| 550px]] ---- == Expansion PCR (Quick test, round2) == * As discussed above, I did a quick run (10ul reaction) with following condition ** Subset A: 100nM template, 15 and 16 cycles ** Subset B: 150nM template, 16 and 17 cycles ** Subset C: 150nM and 200nM. 18 cycles * This time, I used a white real-time PCR tube, so the fluorescent intensity is higher than using a clear tube in the first test. [[File:2014-08-21_test_ExpPCR-90k_15x.PNG| 500px]] [[File:2014-08-21_test_ExpPCR-90k_16x.PNG| 500px]] [[File:2014-08-21_test_ExpPCR-90k_17x.PNG| 500px]] [[File:2014-08-21_test_ExpPCR-90k_18x.PNG| 500px]] * Loaded 5ul of amplicons from each subset at different PCR conditions and also re-loaded amplicons from the 1st test in 6% TBE gel [[File:ZhangLab_2 2014-08-21 18hr 58min_90k-testExpPCR-r2.jpg|500px]] == Expansion PCR in a large volume == * I do expansion PCR of the 3 subset in total volume 300ul for each subset. I use 100nM template for Subset A and B and run for 17 cycles. For subset C, I use 200nM template and run for 18 cycles <u>'''Set A (V6 primers)'''</u> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||9.55||100nM |- | AP1V61U (100uM)||1.20||400nM |- | AP2V6 (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||138.05|| |- | Total||300.00|| |}<br> <u>'''Set B (V4 primers)'''</u> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||9.55||100nM |- | AP1V41U (100uM)||1.20||400nM |- | AP2V4 (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||138.05|| |- | Total||300.00|| |}<br> <u>'''Set C (eMIP_CA1 primers)'''</u> {| {{table}} border =1 | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final conc.''' |- | 3,142.7nM seed oligoes||19.09||200nM |- | eMIP_CA1_F (100uM)||1.20||400nM |- | eMIP_CA1_R (100uM)||1.20||400nM |- | 2x KAPA SYBG fast MM||150.00||1x |- | H2O||128.51|| |- | Total||300.00|| |} * For each subset, I aliquot 50ul to 6X in strip tube 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 18-> 72C 3min -> 15C hold [[File:2014-08-22_ExpPCR_setA-B_17x.PNG| 500px]] [[File:2014-08-22_ExpPCR_setC_18x.PNG| 500px]] * Purified with Qiaquick column (3 columns for each) and elute with EB buffer volume 52ul each --> total volume left = ~140ul * Measure DNA conc. with Nanodrop for each subset * M.W. of dsDNA 148bp = 148bp * 650Da/bp = 96,200 g/mole {| {{table}} class = wikitable | align="center" style="width:180px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''260/230''' | align="center" style="width:100px;background:#f0f0f0;"|'''260/280''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. (nM)''' |- | LMS cluster (set A)||10.83||5.75||2.49||110.87 |- | Selector LMS (set B)||9.46||3.75||2.15||96.85 |- | Selector chr21 (Set C)||13.89||4.17||2.05||142.20 |} * I will give set C to Rui for production PCR * Dilute 1st round amplicons of subset A and subset B to 20nM for using as the template for future production PCR<br> {| {{table}} border = 1 | align="center" style="width:180px;background:#f0f0f0;"|'''Subset''' | align="center" style="width:80px;background:#f0f0f0;"|'''Original conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Add EB buffer to 20nM''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total volume (ul)''' |- | LMS cluster (set A)||110.87||140||636.09||776.09 |- | Selector LMS (set B)||96.85||140||537.95||677.95 |} * Continued to production PCR <br> == Production PCR == * I increase template a little bit from 0.04nM to 0.05 nM since I think that the value from N.D. is from the unamplified template from expansion PCR as well. {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1 rxn''' | align="center" style="width:100px;background:#f0f0f0;"|'''100x rxn mix''' | align="center" style="width:100px;background:#f0f0f0;"|'''Final Conc.''' | align="center" style="width:100px;background:#f0f0f0;"|'''Unit''' |- | 1st round amplicon (20nM)||0.25||25.00||0.05||nM |- | AP1V61U (100uM)||0.40||40.00||400||nM |- | AP2V6 (100uM)||0.40||40.00||400||nM |- | 2x KAPA SYBG fast MM||50.00||5,000.00||1.00||X |- | H2O||49.00||4,900.00|||| |- | Total||100.00||10,005.00|||| |} 95C 30sec -> (95C 10sec -> 55C 20sec-> 72C 30sec) X 16-> 72C 3min -> 15C hold<br> * I used the same condition for subset B but used V4 primers. [[File:2014-08-22_ProductionPCR_setA_14x.PNG| 500px]] [[File:2014-08-22_ProductionPCR_setB_15x.PNG| 500px]] ==== Size selection ==== [[File:ZhangLab_2 2014-08-26 17hr 50min_PAGE-SS_SetA_1.jpg| 300px]] [[File:ZhangLab_2 2014-08-26 17hr 50min_PAGE-SS_SetA_2.jpg| 300px]]<br> <br> [[File:ZhangLab_2 2014-08-26 17hr 50min_PAGE-SS_SetA_3.jpg| 300px]] [[File:ZhangLab_2 2014-08-26 17hr 50min_PAGE-SS_SetA_4.jpg| 300px]]
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