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== PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind9-29) == * I tested if the capture works well by performing qPCR of few samples in small volume 25ul * Sample te be tested: NTC, GL7,GL8 AL-1 and AL3 * Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction) {| {{table}} border =1 | align="center" style="background:#f0f0f0;"|'''Components''' | width="55pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | width="80pt" align="center" style="background:#f0f0f0;"|'''5.2x rxn mix''' |- | Circularized DNA template||5.00||0.00 |- | 10uM AmpF6.4Sol||0.50||2.60 |- | 10uM AmpR6.3Sol||0.50||2.60 |- | 50x SYBG||0.20||1.04 |- | 2x Phusion MM||12.50||65.00 |- | H2O||6.30||32.76 |- | Total||25.00||104.00 |} * Aliquot 20ul, add 5ul of DNA template '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:20120825_testqPCR_BSPP-GL-AL.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | width="60pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | width="80pt" align="center" style="background:#f0f0f0;"|'''45x rxn mix''' |- | Captured template||20.00||0.00 |- | 10uM AmpF6.4Sol||2.00||90.00 |- | 10uM AmpR6.3Indx (X=9-29)||2.00||0.00 |- | 50x SYBG||0.80||36.00 |- | 2x Phusion MM||50.00||2250.00 |- | H2O||25.20||1134.00 |- | Total||100.00||3510.00 |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min (All reactions were stopped at cycle 19) [[File:20120825_qPCR_BSPP-GL-AL.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | GL-1||AmpR6.3Indx9||GL-9||AmpR6.3Indx17||AL-1||AmpR6.3Indx25 |- | GL-2||AmpR6.3Indx10||GL-10||AmpR6.3Indx18||AL-2||AmpR6.3Indx26 |- | GL-3||AmpR6.3Indx11||GL-11||AmpR6.3Indx19||AL-3||AmpR6.3Indx27 |- | GL-4||AmpR6.3Indx12||GL-12||AmpR6.3Indx20||AL-4||AmpR6.3Indx28 |- | GL-5||AmpR6.3Indx13||GL-13||AmpR6.3Indx21||AL-5||AmpR6.3Indx29 |- | GL-6||AmpR6.3Indx14||GL-14||AmpR6.3Indx22|||| |- | GL-7||AmpR6.3Indx15||GL-15||AmpR6.3Indx23|||| |- | GL-8||AmpR6.3Indx16||GL-16||AmpR6.3Indx24|||| |} - Took 80ul of PCR product from replicate1 and 2 (total volume 160ul) - Purified with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl) - Eluted with 60ul EB buffer - Performed PAGE quantification by loading sample 1ul [[File:ZhangLab_2 2012-08-25 21hr 25min_PQ_GL-1.jpg| 500px]] [[File:ZhangLab_2 2012-08-25 21hr 25min_PQ_GL-2.jpg| 500px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' |- | GL-1||10.72||642.91||18.67 |- | GL-2||12.24||734.53||16.34 |- | GL-3||11.98||718.56||16.70 |- | GL-4||12.17||730.11||16.44 |- | GL-5||11.07||664.39||18.06 |- | GL-6||12.74||764.43||15.70 |- | GL-7||10.40||623.89||19.23 |- | GL-8||9.74||584.46||20.53 |- | GL-9||10.88||652.62||18.39 |- | GL-10||11.16||669.83||17.92 |- | GL-11||8.30||497.79||24.11 |- | GL-12||9.40||564.06||21.27 |- | GL-13||9.26||555.74||21.59 |- | GL-14||7.18||430.56||27.87 |- | GL-15||7.37||441.94||27.15 |- | GL-16||8.25||494.83||24.25 |- | AL-1||5.11||306.64||39.13 |- | AL-2||6.38||382.88||31.34 |- | AL-3||5.96||357.37||33.58 |- | AL-4||4.91||294.31||40.77 |- | AL-5||4.45||266.78||44.98 |} - Combine 200ng of each library. -Perform PAGE size selection in 6% TBE 5-well gel (3 gels) [[File:ZhangLab_2 2012-08-26 18hr 46min_PAGE_SS-1.jpg| 500px]] [[File:ZhangLab_2 2012-08-26 18hr 29min_PAGE_SS-2.jpg| 250px]] * From gel images, DNA was overoaded - Resuspended with H2O, total volume 48ul [[File:ZhangLab_2 2012-08-26 22hr 24min_PQ_NP_BSPP_GL-AL.Ind9-29.jpg| 200px]]
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