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==Procedure== ===Calculation of mass of probe required for each human gDNA input=== *Can find calculation on my Google Doc at <https://docs.google.com/spreadsheets/d/10hhMyThBT2nAvxIodQq_1q0RQopnfMlNzSEZEexi0nE/edit?usp=sharing> *For each of the tests, the following is the probe and target masses required: rxn Target (ng) Probe:Target Probe (ng) 1 100ng 500:1 8.25 (my probe designed 5/12, on 100ng) 2 100ng 200:1 3.30 (my probe designed 5/12, on 100ng) 3 100ng 50:1 0.83 (my probe designed 5/12, on 100ng) 4 20ng 1000:1 0.32 (Rui's probe, on 20ng) 5 0ng(NTC, CW) 500:1 8.25 (This is NTC for 20hr probe annealing) 6 0ng(NTC, CW) 200:1 3.30 (This is NTC for 20hr probe annealing) 7 0ng(NTC, CW) 50:1 0.83 (This is NTC for 20hr probe annealing) 8 0ng(NTC, RL) 1000:1 0.32 (This is NTC for 20hr probe annealing) 9 0ng(NTC) ---- 0 (This is full NTC, no template and no probe) --------------------------------------------------------------------------------------------------------------------------------------------------- 10 0ng(NTC, CW) 500:1 8.25 (This is NTC for the 4hr gap-filling and circularization step, to be added AFTER annealing step) 11 0ng(NTC, RL) 1000:1 0.32 (This is NTC for the 4hr gap-filling and circularization step, to be added AFTER annealing step) --------------------------------------------------------------------------------------------------------------------------------------------------- 12 0ng(NTC, CW) 500:1 8.25 (This is NTC for Exo digestion step, to be added AFTER gap-filling and circularization) 13 0ng(NTC, RL) 1000:1 0.32 (This is NTC for Exo digestion step, to be added AFTER gap-filling and circularization) *Dilute my probe mix to 1ng probe/ul (will dilute probes produced on 7/10, which was ~14ul, ~60ng/ul) **Probe concentration is ~60ng/ul -> 1/60d -> 1ul probe + 59ul water (save this for later so will not need to dilute so many times) *Dilute Rui's probe mix to 0.1ng/ul **Probe concentration is ~1ng/ul -> 1/10d -> 2ul probe + 18ul water *Dilute target human gDNA concentration to 10ng/ul **gDNA concentration is ~110ng/ul -> 1/11d -> 4ul gDNA + 40ul water *Final volume of probe and target to add to each rxn rxn Target (ul) Probe (ul) Water (ul) -> to get to 20ul total volume 1 10 8.25 (CW) 1.75 2 10 3.30 (CW) 6.70 3 10 0.83 (CW) 9.17 4 2 3.20 (RL) 14.80 5 0 8.25 (CW) 11.75 6 0 3.30 (CW) 16.70 7 0 0.83 (CW) 19.17 8 0 3.20 (RL) 16.80 9 0 0 20.00 --------------Add after 20hr annealing----------------- 10 0 8.25 (CW) 13.75 (want to get to 22ul total volume because need to mimic the extra 2ul of AmpLigase Buffer) 11 0 3.20 (RL) 18.80 --------------Add after 4hr gap-filling/circularization--------- 12 0 8.25 (CW) 15.75 (want to get to 24ul total volume because need to mimic the extra 2ul SLN mix) 13 0 3.20 (RL) 20.80 ===Probe+Template+Buffer Mix=== *Add 2.00ul of 10x AmpLigase buffer to each reaction tube *Overlay with 40ul mineral oil (can centrifuge in order for oil and solution to separate) *Thermocycler: 95C 30sec -> cool down to 60C at 0.02C/sec -> '''Incubate 60C 20hr''' ===Prep SLN mix solution=== *We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued) *Mastermix recipe Component Stock conc Final conc 1x(ul) MM Vol (13x) (ul) Hemo KlenTaq 100% 20% 0.4 5.2 AmpLigase 5U/ul 0.5U/ul 0.2 2.6 dNTP 1000uM 100uM 0.2 2.6 (MDA dNTP = 25mM, so do 1/25d) 10x AmpLigase Buff 10x 1x 0.2 2.6 H2O 1.0 13 *Thermocycler (continued): Add 2ul SLN to each rxn (1->11) -> '''Incubate 60C 4hr''' -> 94C 2min -> add 2ul ExoI/ExoIII mix (1:1) to each rxn (1->13) -> 37C 2hr -> 94C 5min -> Hold 10C **Note: Right before 4hr is up, prep the ExoI/ExoIII mix at 1:1 ratio ===Thermocycler Program (PP-CAP60 in PCR machine near Noi's bench)=== 1 = 95C 0:30 2 = 94.8C 0:30 -0.2C per cycle 3 = Goto 2, 173 times 4 = 60C Forever (20hr -> add SLN -> 4hr) 5 = 94C 2:00 6 = 37C 2:00:00 7 = 95C 5:00 8 = 10C Forever
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