Chris:LabNotes/FateMapping/Calendar/2014/2014-8-18

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Target capture with padlock probes (designed 5/12) test with Rui's probes as control[edit]

Background[edit]

  • So far, I have not gotten a good probe capture result yet (as the result of capture is usually a small band while mine is currently still a smear on the gel after PCR)
  • One of the suggestions given in the previous lab meeting was the increase the number of controls during each run of the experiments. Because of this, I'll be adding a new negative control to be added in after 20hr annealing but before Hemo KlenTaq gap filling and circularization. I'll also be adding in a reaction for Rui's probe as a positive control
    • Note that for Rui's probes, I'll need to do a 60C annealing. Also, Rui uses 3.2ul of 0.1ng/ul (need to dilute 1/10 from the 1ng/ul aliquot that she gave me) probe for 20ng of gDNA. So, for my experiment, will need to add 16ul of 0.1ng/ul probe for 100ng gDNA. This amounts to a 1000:1 probe:target ratio.
  • Also will try a condition with lower probe:target ratio, as this may be a possibility for the high intensity of the non-specific band.

Procedure[edit]

Calculation of mass of probe required for each human gDNA input[edit]

    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[edit]

  • 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[edit]

  • 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)[edit]

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