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
  • 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]

    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