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==Test 5 different polymerases for capture with duplicates == * Testing 5 different polymerases with varied input amounts. === Capture setup === {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''Converted sample''' | align="center" style="background:#f0f0f0;"|'''Amount sample (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume sample (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume probes (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume of 10x ampligase buffer''' | align="center" style="background:#f0f0f0;"|'''Volume water (uL)''' | align="center" style="background:#f0f0f0;"|'''Total volume''' |- | HT-A-56.4-HemoKlenTaq_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-HemoKlenTaq_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |- | HT-A-56.4-KlenTherm_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-KlenTherm_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |- | HT-A-56.4-NoPolymerase_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-NoPolymerase_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |- | HT-A-56.4-PfuTurboCX_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-PfuTurboCX_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |- | HT-A-56.4-Stoffel_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-Stoffel_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |- | HT-A-56.4-TruncatedTaq_1||HT1080-1||100.0||2.08||2.70||2.00||13.22||20.00 |- | HT-A-56.4-TruncatedTaq_2||HT1080-1||200.0||4.16||2.70||2.00||11.14||20.00 |} * Make two master mixes: 200 ng master mix: 24.95 uL converted HT1080 (48.1 ng/uL) 27.36 uL set A probes (13.6 ng/uL) 12.00 uL 10x Ampligase buffer 55.69 uL nuclease-free water 100 ng master mix: 12.47 uL converted HT1080 13.68 uL set A probes 12.00 uL 10x Ampligase buffer 93.85 uL nuclease-free water * Aliquot 20 uL into each labeled 200 uL PCR tube. * Spin down reaction & layer on top with mineral oil. ===Capture program=== * Same as previous run. ==="S"LN set up=== * Add "S"LN mix at 20 hour 56.4C incubation. * For PfuTurboCX Hotstart, incubate an aliquot (3 uL) at 95C for 3 minutes. * Use 3 U of each polymerases just like in previous run. I don't want to use too much as some of these polymerase samples have very limited amount & they are expensive. * Make LN master mix (14X): 1.4 uL Ampligase (5U/ul) 3.5 uL dNTP (1 mM) 3.5 uL 10x Ampligase buffer * Aliquot 1.5 uL to 6 tubes. Stoffel (ST) + 0.75 uL ST + 4.00 uL H2O + 1.5 uL LN mix HemoKlentaq (HK) + 0.75 uL HK + 4.00 uL H2O + 1.5 uL LN mix KlenTherm (KT) + 0.75 uL KT + 4.00 uL H2O + 1.5 uL LN mix PfuTurboCx (PT) + 3.00 uL PT + 1.75 uL H2O + 1.5 uL LN mix Truncated Taq(TT)+ 1.50 uL TT + 3.25 uL H2O + 1.5 uL LN mix No-Pol (NP) + 4.75 uL H2O + 1.5 uL LN mix * Add 2.5 uL SLN to corresponding labeled capture reaction. ===Capture PCR=== * Reaction: 1x 14x DNA template 4.00 uL -- PhusionFlash, 2xMM 20.00 uL 280.00 uL AmpF6.4Sol, 10uM 0.80 uL 11.20 uL AmpR6.3Ind22, 10uM 0.80 uL 11.20 uL SybrGreen, 50x 0.16 uL 2.24 uL Nuclease free water 14.24 uL 199.36 uL * PCR program: 98C (10s) -> [ 98C (10s) -> 58C (30s) -> 72C (20s) ] - 8 cycles -> [ 98C (10s) -> 72C (20s) ] - 22 cycles -> 72C (3 min) * qPCR plots [[File:HOTSPOTS_E_capture_test_polymerases_2_100ng_qPCR.bmp |500px]][[File:HOTSPOTS_E_capture_test_polymerases_2_200ng_qPCR.bmp|500px]] * PAGE gels [[File:HOTSPOTS_E_capture_test_polymerases_2_100ng.jpg |300px]][[File:HOTSPOTS_E_capture_test_polymerases_2_200ng.jpg|300px]] ===Discussion=== * The most intense bands are from PfuTurbo Cx and Stoffel. Although the results from HemoKlentaq looks good, but required more PCR cycles. KlenTherm also worked, I might have messed up while adding SLN to the 100 ng Klentherm and both the Truncated Taq reactions. But those were not the best performing polymerases in all the tests so far. Stoffel have the least background as well, which might be a good indication that it would result in better mapping rates. * Although PfuTurbo have very good amplification, I am concerned about the higher background with PfuTurboCx in between 200-400 bp.
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