Chris:LabNotes/FateMapping/Calendar/2014/2014-6-12
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Target capture with padlock probes (designed 5/12) test[edit]
Background[edit]
- 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
- Probe:Target ratio = 200:1, 300:1, 400:1
- 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[edit]
Calculation of mass of probe required for each human gDNA input[edit]
- 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[edit]
- 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[edit]
- 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)[edit]
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[edit]
- For the Probe+Template+Buffer Mix step, need to incubate at 55C 20hr BEFORE adding SLN then would incubate for another 4hr