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=Target capture with padlock probes (designed 5/12) test= ==Background== *Over the past few weeks, I've made ~1ug of padlock probes using Noi's probe production protocol (details to be posted soon) *Today, we want to test these probes (designed 5/12) on capture efficiency of human genomic DNA provided by Alan (GM12878 P21 [5/23/14] 148ng/ul) *The overall experimental design today will be testing the target capture using three probe:target ratios and three human gDNA input amounts **Probe:Target ratio = 200:1, 300:1, 400:1 ***This was based on Noi's recommendation that we may need to use a higher probe:target ratio because only have ~9000 unique probes designed in the oligo mix **Target input amount = 50ng, 100ng, 200ng ***This is in order to obtain a comparable result to Fluidigm's C1 output of ~100ng of MDA product *In the future, if this experiment works, this will optimize most the Probe:Target ratio necessary for successful capture so we will be able to move on to capture of in-tube MDA amplicons ==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 50ng 1:200 1.65 2 50ng 1:300 2.48 3 50ng 1:400 3.30 4 100ng 1:200 3.30 5 100ng 1:300 4.95 6 100ng 1:400 6.60 7 200ng 1:200 6.60 8 200ng 1:300 9.90 9 200ng 1:400 13.20 *Dilute probe mix to 2ng probe/ul **Probe concentration is ~50ng/ul -> 1/25d -> 1ul probe + 24ul water *Dilute target human gDNA concentration to 20ng/ul **gDNA concentration is ~50ng/ul -> 1/2.5d -> 20ul gDNA + 30ul water) *Final concentration of probe and target to add to each rxn rxn Target (ul) Probe (ul) Water (ul) -> to get to 20ul total volume 1 2.5 0.825 16.675 2 2.5 1.24 16.26 3 2.5 1.65 15.85 4 5.0 1.65 13.35 5 5.0 2.48 12.525 6 5.0 3.30 11.7 7 10.0 3.30 6.7 8 10.0 4.95 5.05 9 10.0 6.60 3.4 ===Probe+Template+Buffer Mix=== *Add 2.00ul of 10x AmpLigase buffer to each reaction tube *Thermocycler: 95C 30sec -> cool down to 55C at 0.02C/sec -> add SLN mix solution ===Prep SLN mix solution=== *We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued) *Mastermix recipe Component Stock conc Final conc MM Vol (ul) Hemo KlenTaq 100% 20% 6.00 AmpLigase 5U/ul 0.5U/ul 3.00 dNTP 1000uM 100uM 3.00 (MDA dNTP = 25mM, so do 1/25d) 10x AmpLigase Buff 10x 1x 3.00 H2O 15.00 *Thermocycler (continued): Add 2ul SLN to each rxn -> 55C 4hr -> 94C 2min -> add ExoI/ExoIII mix (1:1) -> 37C 2hr -> 94C 5min -> Hold 4C **Note: Right before 4hr is up, prep the ExoI/ExoIII mix at 1:1 ratio ===Thermocycler Program (KZ-CPG)=== 1 = 95C 0:30 2 = 94.8C 0:30 -0.2C per cycle 3 = Goto 2, 193 times 4 = 55C forever (20hr -> add SLN -> 4hr) 5 = 94C 2:00 6 = 37C 2:00:00 7 = 95C 5:00 8 = 4C Forever ==Results== *For the Probe+Template+Buffer Mix step, need to incubate at 55C 20hr BEFORE adding SLN then would incubate for another 4hr
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