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==Capture== Set up capture reaction in low profile strip tubes (or PCR tubes/plates). Spin down the reaction mix and add 1-2 droplets of mineral oil with P200. Use Microseal film to cover the tubes. {| {{table}} | align="left" style="background:#f0f0f0;"|'''Probe/Target Ratio'''|| 100|| |- | align="left" style="background:#f0f0f0;"|'''Probes size'''||220000|| |- | align="left" style="background:#f0f0f0;"|'''Template'''||300||ng |- | align="left" style="background:#f0f0f0;"|'''Human gDNA MW'''||1.82E+012||g/mole (3E9bpx607.4D/bp+157.9D) |- | align="left" style="background:#f0f0f0;"|'''Human gDNA '''||1.65E-019||moles |- | align="left" style="background:#f0f0f0;"|'''Probes'''||1.65E-017||moles |- | align="left" style="background:#f0f0f0;"|'''Probes MW'''||6.88E+9||g/mole (220000x103bpx303.7D/bp+79D) |- | align="left" style="background:#f0f0f0;"|'''Amount probes required'''||113||ng |- | |||| |- | align="left" style="background:#f0f0f0;"|'''Concentration probes 1'''||27||ng/ul |- | align="left" style="background:#f0f0f0;"|'''Vol||4.20||ul |- | align="left" style="background:#f0f0f0;"|'''Concentration probes 2'''||23||ng/ul |- | align="left" style="background:#f0f0f0;"|'''Vol'''||4.93||ul |} '''Reaction Mix''' {| {{table}} border =1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Vol(ul)''' | align="center" style="background:#f0f0f0;"|'''Probes(ul)''' | align="center" style="background:#f0f0f0;"|'''10xAmpligase Buffer (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | Sarven, 499T (biscvt)||91.5||3.28||4.2||1||1.53||10 |- | Sarven, 499N (biscvt)||67.9||4.42||4.2||1||0.39||10 |- | Sarven, Jurkat (biscvt)||212.5||1.41||4.2||1||3.39||10 |- | Dinh, 536T (biscvt)||76.2||3.94||4.9||1||0.16||10 |- | Dinh, 536N (biscvt)||105.4||2.85||4.9||1||1.25||10 |- | Dinh, Jurkat (biscvt)||212.5||1.41||4.9||1||2.69||10 |} '''Program''' 95c 30sec -> cool down to 58C at 0.02C/sec -> 58C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 58C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold. '''Adding 2ul SLN mix after >20 h of 58C incubation:''' Leave tubes in thermocycler and just remove Microseal film cover to add SLN mix. Use P10, low retention tips and make sure to reach the bottom of the tube before dispensing. Dispense slowly, then slowly pull out the pipette tip before releasing hold on dispenser. Ensure that all reagents were pumped out. Cut new Microseal films to cover the tubes. '''Adding 2ul ExoI/III mix after 3-4 h of 58C incubation:''' Perform same steps as adding SLN. {| {{table}} border="2" | align="center" style="background:#f0f0f0;"|'''SLN Mix''' | align="center" style="background:#f0f0f0;"|''' 1x''' | align="center" style="background:#f0f0f0;"|'''16x''' | align="center" style="background:#f0f0f0;"|'''Final Conc''' |- | H2O||0.55||8.8|| |- | dNTP(1mM)||0.05||0.8||50uM |- | 10xAmpligase Buffer||0.1||1.6||1x |- | AmpLigase||0.1||1.6||0.5U/ul |- | Stoffel||0.2||3.2||2U/ul |- | TOTAL||1||16|| |- |} '''For ExoI/III mix, add equal volumes of Exo I and Exo III.'''
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