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==Test 5 different polymerases for capture == * Due to the high background from all my previous captures, I need to further optimize my captures. Also, it's important to have a very low failure rate for the capture in order to save precious Tet1-treated DNA. * For this test, I will continue to use the Ampligase buffer. If the polymerases do not work well in Ampligase buffer, I will switch to a different buffer next. ** If using a different PCR buffer, I need to add NAD for Ampligase. * Also only subset E is used in this capture at 200:1 ratio. === 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''' |- | (HK) HT-E-56.4-HemoKlenTaq||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |- | (PT) HT-E-56.4-PfuTurboCX||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |- | (ST) HT-E-56.4-Stoffel||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |- | (KT) HT-E-56.4-KlenTherm||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |- | (TT) HT-E-56.4-TruncatedTaq||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |- | (-) HT-E-56.4-NoPolymerase||HT1080-1||300.0||7.21||2.70||2.00||8.09||20.00 |} ===Capture program=== 1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 192 times -> 56.4°C 3. 56.4°C 20h ADD 2.5 uL Pol-LN mix 4. 56.4°C 20h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix 6. 37°C 2h 7. 90°C 5m 8. 4°C forever ==="S"LN set up=== * For a final volume of 2.5 uL to each tube: 0.1 uL of 5 U/uL Ampligase (final = 0.5 U/uL), 0.25 uL of 1 mM dNTPs, 0.25 uL of 10X Ampligase Buffer. I added 0.60 uL of LN mix to each first. * Added LN mix at 20 hour 56.4C incubation. * For PfuTurboCX Hotstart, I aliquoted 1.5 uL that was then incubated at 95C for 3 minutes. Then transferred 1.2 uL to the capture reaction, (tube - PT). * I will use 3 U of each polymerases: {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Polymerase''' | align="center" style="background:#f0f0f0;"|'''Concentration (U/ul)''' | align="center" style="background:#f0f0f0;"|'''3 U Vol (uL)''' | align="center" style="background:#f0f0f0;"|'''Water (uL)''' |- | Stoffel||10||0.3||1.6 |- | HemoKlentaq||5||0.6||1.3 |- | KlenTherm||10||0.3||1.6 |- | PfuTurboCX||2.5||1.2||0.7 |- | Truncated Taq||5||0.6||1.3 |- | No-Polymerase||0||0||1.9 |} * After adding polymerases, I added water last. * After 20 hours, denature DNA at 95C for 3 minutes. '''The polymerases and Ampligase do not get heat inactivated easily.''' * Add 1 uL of Exo I and 1 uL of ExoIII. Incubate at 37C for 2 hours. * After ExoI/III, I performed amplification with Phusion Flash and saw a big smear in all my lanes. I believe this is due to the ExoI/III reaction, so I repeated ExoI&III digestion with new tubes of enzymes & repeated amplification. ===Capture PCR=== * 1X Reaction: captured circles 4.00 uL (half of original capture+18uL water+1 uL ExoI + 1 uL ExoIII) PhusionFlash (2X) 20.00 uL AmpF6.4Sol (10uM) 0.80 uL AmpR6.3Indx12(10uM) 0.80 uL SybrGreen (50x) 0.16 uL Water 14.24 uL * PCR program: 98C (10s) -> [ 98C (10s) -> 58C (30s) -> 72C (20s) ] - 8 cycles -> [ 98C (10s) -> 72C (20s) ] - 22 cycles -> 72C (3 min) * qPCR plot: [[File:HOTSPOTS_E_capture_test_polymerases_1_qPCR.bmp | 500px]] * TBE PAGE gel: (expected band at 471 bp) [[File:HOTSPOTS_E_capture_test_polymerases_1.jpg | 300px]] ===Discussion=== * All 5 polymerases worked in Ampligase buffer but they vary significantly. I plan to repeat the experiment using duplicates (but vary input: 100ng and 200ng) to confirm that these differences are real. * I plan to move capture reaction onto ice when adding ExoI/III. I believe that when the reaction cools to 37C on the thermocycler, there may be an opportunity for Ampligase to ligase unused probes together. After discussing with others in the lab, we wonder if Ampligase may still have activity after incubation for long periods at ~55C, if it does, then adding ExoI/III on ice may be crucial to reducing background levels. * I will continue performing capture PCR with Phusion Flash as this mix seems to reduce the background signals between 200-400bp. * Since the concentration of HemoKlentaq is not known, we usually assume it to be 10U/uL and use 5 U (0.5 uL). For this experiment, I assumed the concentration to be 5 U/uL, and used 3 U (0.6 uL). The amount of HemoKlentag that I used is very close to the usual amount.
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