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=== 2nd round PCR === ==== OPTIMIZE PCR cycle number ==== * I optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul) and stopped reaction at 10X, 12X, 14X, and 16X cycles. * Then analyze in 12% TBE gel * Select the optimum cycle number to amplify the rest of 1st round PCR <u>'''PC-P smaple'''</u> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''13 rxn mix''' |- | Purified 1st round DNA||||||||||12.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||8.00||104.00 |- | dNTP mix||10||mM||0.25||mM||1.00||13.00 |- | TruS_F/R||10||uM||0.3||uM||1.20||15.60 |- | 50X SYBR||50||X||0.4||X||0.32||4.16 |- | Phusion HF||2||unit/ul||||||0.80||10.40 |- | H2O||||||||||16.68||216.84 |- | Total||||||||||40.00|| |} * Note: From the condition above, I used Phusion HF 1ul (2units) in 50ul reaction (normalized to 50ul reaction), which is too high. In the following steps, I reduced it to 0.5ul (1unit) in 50ul reaction. :- Aliquot 28 + 12ul DNA template :- Mix well. Split 4X of 8ul ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [10X, 12X, 14X, 16X] cycles --> 72C for 2min''<br> :- Added 10ul 2X loading dye to each strip tube & mix well :- Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TT-10X.jpg| 500px]] [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-12X.jpg| 480px]] * Note: I did a wrong labeling for 12 cycles of PC-P samples. They should be P in stead of N [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-14X.jpg| 495px]] [[File:ZhangLab_2 2014-06-23 11hr 45min_1ngRRBS_2nd_TEST-16X.jpg| 490px]] * 12-16 cycles are over-amplified for PC-P samples. Even 10X cycles is still too high intensity compare to some examples of the reference. * Did the same way for NC-P samples, but only monitor at 8X and 9X. However, I looked at qPCR curve too late, so I could only stopped at 9X and 10X cycles. [[File:ZhangLab_2 2014-06-23 16hr 09min_NC_1ngRRBS_2nd_TEST_9X.jpg| 500px]] [[File:ZhangLab_2 2014-06-23 16hr 09min_NC_1ngRRBS_2nd_TEST_10X.jpg| 500px]] * N10 & N11 did not work!! * These steps may spend a lot of time and gels. However, in the future, I do not need to do this optimization as I would know roughly number of cycle to stop at. * The reason that I need to look at the gel image first is that the adaptor dimers level from ligation reaction is very hight (no purification before bisulfite conversion). Therefore, the qPCR curve of NTC and the samples with DNA are not different. * From this optimization, I know that I should stop before qPCR curve reaches the plateau as the gel intensity is consistent with the qPCR curve. Letting PCR go after it reaches the plateau causing over-amplification and a smear a long the lane instead of a smear at the range between ~160-360bp * I would do 8 cycles for the rest of 1st round PCR for both PC-P and NC-P samples but do 9 cycles for sample PC-P-10 as it had very low intensity. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''18.5 rxn mix''' |- | Purified 1st round DNA||||||||||25.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||20.00||370.00 |- | dNTP mix||10||mM||0.25||mM||2.50||46.25 |- | TruS_F/R||10||uM||0.3||uM||3.00||55.50 |- | 50X SYBR||50||X||0.4||X||0.80||14.80 |- | Phusion HF||2||unit/ul||||||1.00||18.50 |- | H2O||||||||||47.70||882.45 |} :- Aliquot 75ul, add 25ul 1st round PCR :- Mix well and split 48ul, 2X. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [8X, 9X for PC-P-10] cycles --> 72C for 2min''<br> [[File:ZhangLab_2 2014-06-23 20hr 02min_1ngRRBS_PC-P_PQ-3ulof55.jpg| 500px]] [[File:ZhangLab_2 2014-06-23 20hr 02min_1ngRRBS_NC-P_PQ-3ulof55.jpg| 450px]] ==== PAGE quantification results ==== <u>PC-P samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | PC-P-1||1||7.12||356.17 |- | PC-P-2||2||8.09||404.41 |- | PC-P-3||4||10.82||540.90 |- | PC-P-4||5||11.25||562.37 |- | PC-P-5||6||7.49||374.73 |- | PC-P-6||7||8.61||430.26 |- | PC-P-7||8||10.23||511.64 |- | PC-P-8||10||7.87||393.59 |- | PC-P-9||11||3.25||162.28 |- | PC-P-10||12||1.94||96.97 |} <u>NC-P samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | NC-P-1||13||4.82||241.12 |- | NC-P-2||14||7.60||380.06 |- | NC-P-3||15||9.37||468.59 |- | NC-P-5||16||3.86||192.81 |- | NC-P-6||27||6.31||315.29 |- | NC-P-7||18||2.62||131.19 |- | NC-P-8||19||3.76||187.99 |- | NC-P-9||20||4.61||230.44 |} ==== Library pooling ==== * To avoid index duplication, I need to split PC-P and NC-P libraries into 2 pools for each :- <span style="color:crimson">(10M each) NP-RRBS-CTT-5ng, Jun3: <u>Index</u> 9, 10, 11, 13</span> :- <span style="color:crimson">(20M each) NP-RRBS-CTT-100ng, Jun13: <u>Index</u> 18, 19, 20, 21</span> <br> :- <span style="color:green">(20M each) NP-RRBS-PCT-100ng-Jun16: <u>Index</u> 1, 2, 4, 5, 6, 7, 8, 12, 14, 15</span> --> ''(This pool should take the whole lane. Will not be mixed with other pool)'' <br> :- <span style="color:crimson">(4.5M each) NP-RRBS-PC-P-1ng-p1-Jun20: <u>Index</u> 1, 2, 4, 5, 6, 7, 8, 12</span> :- <span style="color:blue">(4.5M each) NP-RRBS-PC-P-1ng-p2-Jun20: <u>Index</u> 10, 11</span> --> MiSeq <br> :- <span style="color:crimson">(4.5M each) NP-RRBS-NC-P-1ng-p1-Jun20: <u>Index</u> 14, 15, 16, 27</span> :- <span style="color:blue">(4.5M each) NP-RRBS-NC-P-1ng-p2-Jun20: <u>Index</u> 13, 18, 19, 20</span> --> MiSeq NP-RRBS-PC-P-1ng-p1-Jun20 {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 120ng (ul)''' |- | PC-P-1||1||7.12||16.85 |- | PC-P-2||2||8.09||14.84 |- | PC-P-3||4||10.82||11.09 |- | PC-P-4||5||11.25||10.67 |- | PC-P-5||6||7.49||16.01 |- | PC-P-6||7||8.61||13.94 |- | PC-P-7||8||10.23||11.73 |- | PC-P-10*||12||1.94||50.00 |} NP-RRBS-PC-P-1ng-p2-Jun20 {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 90ng (ul)''' |- | PC-P-8||10||7.87||11.43 |- | PC-P-9||11||3.25||27.73 |} NP-RRBS-NC-P-1ng-p1-Jun20 {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | NC-P-2||14||7.60||13.16 |- | NC-P-3||15||9.37||10.67 |- | NC-P-5||16||3.86||25.93 |- | NC-P-6||27||6.31||15.86 |} NP-RRBS-NC-P-1ng-p2-Jun20 {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 100ng (ul)''' |- | NC-P-1||13||4.82||20.74 |- | NC-P-7||18||2.62||38.11 |- | NC-P-8||19||3.76||26.60 |- | NC-P-9||20||4.61||21.70 |} ==== PAGE size selection ==== [[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_PC-P_p1-p2.jpg| 400px]] PC-P pool1: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification PC-P pool2: Resuspend with 15ul H2O x 1tubes --> volume 15ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_NC-P_pool1.jpg| 300px]] NC-P pool1: Resuspend with 12ul H2O x 2tubes --> volume 24ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-06-24 11hr 14min_PAGE-SS_NC-P_pool2.jpg| 400px]] NC-P pool2: Resuspend with 10ul H2O x 3tubes --> volume 30ul, 4ul for PAGE verification ==== PAGE verification ==== [[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]]
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