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=== 2nd round PCR === '''OPTIMIZE PCR cycle number''' * I only TEST 9 cycles of PCR {| {{table}} border = 1 | align="center" style="width:140px;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;"|'''26 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||52.00 |- | dNTP mix||10||mM||0.25||mM||0.25||6.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||7.80 |- | 50X SYBR||50||X||0.4||X||0.08||2.08 |- | Phusion HF||2||unit/ul||||||0.10||2.60 |- | H2O||||||||||4.27||111.02 |- | Total||||||||||10.00|| |} :- Aliquot 7 + 3ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 9X 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 2014-07-02 14hr 25min_1ngRRBS_6P_1-10_TEST-9x.jpg| 500px]] [[File:ZhangLab_2 2014-07-02 14hr 25min_1ngRRBS_7P_1-10_TEST-9x.jpg| 500px]] * A smear for sample 6P_1-10 did not look very good for me since it 's supposed to be very sharp compare to previous experiment. I think it might be over-amplified. That's why there are s smear above the expected ranged. * I think 8 cycles should reduce background and increase the intensity of a smear between 150-350bp. However, I'm afraid to amplified in all of template DNA at once. I do amplification 1Xrxn of 50ul instead of 2X of 50ul as usual. {| {{table}} border = 1 | align="center" style="width:140px;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;"|'''20.5 rxn mix''' |- | Purified 1st round DNA||||||||||15.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||10.00||205.00 |- | dNTP mix||10||mM||0.25||mM||1.25||25.63 |- | TruS_F/R||10||uM||0.3||uM||1.50||30.75 |- | 50X SYBR||50||X||0.4||X||0.40||8.20 |- | Phusion HF||2||unit/ul||||||0.50||10.25 |- | H2O||||||||||21.35||437.68 |- | Total||||||||||50.00|| |} :- Aliquot 35 + 15ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8X cycles --> 72C for 2min''<br> :- I load 5ul of amplified DNA at 8 cycles [[File:ZhangLab_2 2014-07-02 21hr 55min_6P_1-10_8x_50ulrxn.jpg|500px]] [[File:ZhangLab_2 2014-07-02 21hr 55min_7P_1-10_8x_50ulrxn.jpg|500px]] * The result looked better than 9 cycles since I can see less background and the border of a smear for samples 6P_1-10 look sharper.Then I continue to do another 50ul PCR using the same condition above. ==== NOTE ==== * Optimization of PCR cycles in small volume (10ul) is still VERY IMPORTANT for RRBS (2-3 cycles different, like 8X cycles and 10X cycles in 2nd round PCR for 1-5ng input) to get a good result even we know roughly cycles # for different input amount. :- I pool ~80ul PCR product from 1st and 2nd times and elute with 55ul EB buffer :- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries <u>'''6-P_1-10: Plasma sample from colon cancer patients'''</u> [[File:ZhangLab_2 2014-07-03 17hr 22min_PQ_6Prrbs_1-10_3ul.jpg| 500px]] <u>'''7-P_1-10: Plasma sample from lung cancer patients'''</u> [[File:ZhangLab_2 2014-07-03 17hr 22min_PQ_7Prrbs_1-10_3ul.jpg| 500px]] ==== PAGE quantification results ==== <u> 006-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)''' |- | 6-P-1||13||5.18||258.95 |- | 6-P-2||14||4.84||242.15 |- | 6-P-3||15||3.18||159.17 |- | 6-P-4||16||3.82||191.13 |- | 6-P-5||27||2.07||103.50 |- | 6-P-6||18||1.85||92.26 |- | 6-P-7||19||3.04||151.94 |- | 6-P-8||20||2.88||143.84 |- | 6-P-9||21||4.71||235.50 |- | 6-P-10||22||2.59||129.26 |} <u> 007-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)''' |- | 7-P-1||1||7.11||355.52 |- | 7-P-2||2||5.49||274.61 |- | 7-P-3||4||3.93||196.40 |- | 7-P-4||5||8.14||407.01 |- | 7-P-5||6||3.26||163.24 |- | 7-P-6||7||5.83||291.54 |- | 7-P-7||8||8.10||405.02 |- | 7-P-8||10||9.79||489.42 |- | 7-P-9||11||3.71||185.74 |- | 7-P-10||12||6.48||324.06 |}<br> ==== <u>Library pooling</u> ==== =====<span style="color:mediumblue"><u> 006-P samples</u></span>===== <span style="color:mediumblue">NP-RRBS-6P_1ng-Jun29</span> {| {{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 75ng (ul)''' |- | 6-P-1||13||5.18||14.48 |- | 6-P-2||14||4.84||15.49 |- | 6-P-3||15||3.18||23.56 |- | 6-P-4||16||3.82||19.62 |- | 6-P-5||27||2.07||36.23 |- | 6-P-6||18||1.85||40.65 |- | 6-P-7||19||3.04||24.68 |- | 6-P-8||20||2.88||26.07 |- | 6-P-9||21||4.71||15.92 |- | 6-P-10||22||2.59||29.01 |} :- Add 50ul of 6X loading dye and load 73.93 in 4 well of 5-well 6% TBE gel =====<span style="color:red"><u> 007-P samples</u></span>===== <span style="color:red">NP-RRBS-7P_1ng-Jun29</span> {| {{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)''' |- | 7-P-1||1||7.11||16.88 |- | 7-P-2||2||5.49||21.85 |- | 7-P-3||4||3.93||30.55 |- | 7-P-4||5||8.14||14.74 |- | 7-P-5||6||3.26||36.76 |- | 7-P-6||7||5.83||20.58 |- | 7-P-7||8||8.10||14.81 |- | 7-P-8||10||9.79||12.26 |- | 7-P-9||11||3.71||32.30 |- | 7-P-10||12||6.48||18.52 |} :- Add 50ul of 6X loading dye and load 67.31 in 4 well of 5-well 6% TBE gel<br> ---- ==== PAGE size selection ==== * I forgot to take the picture of PAGE size-selection :- NP-RRBS-6P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification :- NP-RRBS-7P_1ng-Jun29: Resuspend with 12ul H2O x 4tubes --> volume 48ul, 4ul for PAGE verification ==== PAGE verification ====
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