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==Helping Dheeraj to test Klentaq capture reactions== *Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with Noi to test the protocol, but Noi is sick and I will be helping to perform the qPCR *They have 12 samples and we will be amplifying the captured samples with qPCR using the same protocol as it in Stoffol capture *1-6 done in condition 16 hours incubation 4 hours polymerization *the second set we did the same 20 hours incubation and 20 hours polymerization ==Protocol== ==PCR amplification to check if capture works== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" valign="bottom" | width="137" height="15" | Components | width="65" | 1x rxn (ul) | width="65" | 12x rxn (ul) | width="65" | Final Conc. |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Circularized DNA template | align="center" | 2.5 | align="center" | 0 | align="center" | |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | KAPA SSYBR FAST MM 2X | align="center" | 12.5 | align="center" | 150 | 1X |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | 10uM AmpF6.4Sol | align="center" | 0.5 | align="center" | 6 | 200nM |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | 10uM AmpR6.3Ind23 | align="center" | 0.5 | align="center" | 6 | 200nM |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | H2O | align="center" | 10 | align="center" | 120 | align="center" | |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Total | align="center" | 25 | align="center" | | align="center" | |} *98C 30sec *(98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles *(98C 10sec -> 72C 20sec) X 10-15 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau) *72C 3min *Hold 15C ==qPCR results== [[File:01022014_1.JPG|400px|16Hr,4Hr]] [[File:01022014_2.JPG|400px|20Hr,20Hr]] *L:16Hr, 4Hr R:20Hr, 20Hr *For 16Hr,4Hr set we should stop at 8 cycles *For 20Hr, 20Hr set we should stop the amplification after 7 cycles ==PAGE Verification== [[File:ZhangLab_2 2014-01-02 15hr 41min.jpg|400px|PAGE]] ==PCR== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt;font-weight:bold" valign="bottom" | width="137" height="15" | Components | width="65" | 1x rxn (ul) | width="65" | 12x rxn (ul) | width="65" | Final Conc. |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Circularized DNA template | align="center" | 10 | align="center" | 0 | align="center" | |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | KAPA SSYBR FAST MM 2X | align="center" | 50 | align="center" | 600 | 1X |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | 10uM AmpF6.4Sol | align="center" | 2 | align="center" | 24 | 200nM |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | 10uM AmpR6.3IndX | align="center" | 2 | align="center" | 0 | 200nM |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | H2O | align="center" | 36 | align="center" | 432 | align="center" | |- style="font-size:12pt" valign="bottom" |style="font-weight:bold" height="15" | Total | align="center" | 100 | align="center" | | align="center" | |} *98C 30sec *(98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles *(98C 10sec -> 72C 20sec) X 10 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau) *72C 3min *Hold 15C *Used Index #1-12 for amplification [[File:01022014_16hr_4hr.JPG|400px]] [[File:01022014_20hr_20hr.JPG|400px]] *L:16Hr, 4Hr R:20Hr, 20Hr ==AMPure bead purification protocol== *Mix 1 volume of bead with PCR product by pipetting 10X *Leave at RT for 8min *Sit on magnetic 96-ring stand for 5min *Wash twice with freshly prepared 200ul 80% EtOH (after adding 80% EtOH, wait for 30sec before pipette up and down few times and discard spnt, and avoid to touch the beads) *Dry the beads for 3 min (do not overdry) *Resuspend the bead with 50ul EB buffer *Leave at RT for 2 min *Sit on magnetic 96-ring stand for 3-5min *Transfer elute DNA to new tube ==PAGE Gel Size Selection==
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