Chris:LabNotes/FateMapping/Calendar/2015/2015-10-23

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Target Capture - Using C1 amplicons and extended MDA from WZTO CBL) w/ 9/22/2014 probes (Hemo and Phusion Reactions) (Part II)[edit]

Background[edit]

File:Cw 20151022 C1 WZTO CBL.pdf
     1     2     3     4     5     6     7     8     9     10     11     12
A    ---------------------------------1------------------------------------
B                                     |
C                                     V
D    ---------C1 Amplicons-----  2NTC  ---------C1 Amplicons----------  2NTC
E                                     |
F                                     |
G                                     V
H    ---------------------------------8-------------------------------------
     ----------Hemo-------------------|-----------------Phusion-------------

Procedure[edit]

Prep KLN mix solution (6 strips, 8 rxn's each)[edit]

  • We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued)
  • First, perform the following dilutions:
    • Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt)
  • Mastermix recipe
Component            Stock conc     Final conc     1x(ul)     MM Vol (54x) (ul)     
Hemo KlenTaq         100%           20%            0.4        21.6
AmpLigase            5U/ul          0.5U/ul        0.2        10.8
dNTP                 1mM            100uM          0.2        10.8
10x AmpLigase Buff   10x            1x             0.2        10.8
H2O                                                1.0        54
  • Aliquot 13ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions
  • Add 2ul KLN mix to all strips (columsn 1->6) using multichannel

Prep Phusion mix solution (6 strips, 8 rxn's each)[edit]

  • First, perform the following dilutions:
    • Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) (same as above)
    • Dilute NAD+ 1/10d (45ul NAD+ + 405ul H2O)(50mM -> 5mM)
  • Mastermix recipe
Component              Stock conc     Final Amount     1x(ul)              MM Vol (53x) (ul)
NAD+                   5mM            40nmol           8.0 (after dil)     424
dNTP                   1mM            600pmol          0.6 (after dil)     31.8
Betaine                5M             15umol           3.0                 159
10x AmpLigase Buff     10x            1x               2.0                 106
AmpLigase              5U/ul          10U              2.0                 106
Phusion HF DNA Pol     2000U/ml       6.4U             3.2                 169.6
Water                                                  1.2                 63.6
  • Aliquot 130 Phusion mix to each well of a strip tube so can use multichannel when adding to the reactions
  • Add 20ul Phusion mix to all strips (columns 7->12) using multichannel
  • Thermocycler (continued): Add KLN or Phusion mix to each rxn -> Incubate 60C 4hr -> 94C 2min
    • Note: I took out tubes immediately after 94C incubation was done and put on ice for minute then add Exo mix and vortex/centrifuge then put back in thermocycler for 37C 2hr incubation

ExoI/ExoIII treatment[edit]

  • We want to then add 20U ExoI and 100U ExoIII
  • Mastermix recipe
Component     Stock Conc     Final Amount     1x(ul)      MM Vol (116x) (ul)
ExoI          20U/ul         20U              1           116
ExoIII        200U/ul        100U             0.5         58
Water                                         0.5         58
  • Aliquot 28ul Exo mix to each well of a strip tube so can use multichannel when adding to the reactions
  • Thermocycler (continued): Add 2ul ExoI/ExoIII mix -> 37C 2hr -> 95C 5min

Phusion PCR[edit]

  • We are going to use the target captured circularized DNA as template for Phusion reaction
  • Note: Since we are not going to do qPCR, we don't need to add SYBR into rxn mix
  • Mastermix recipe (we'll need to decrease the PCR volume because of the higher number of reactions required and limited reagents we have on-hand; to make things simple, we'll use the same input template volume into each PCR reaction)
Component                     1x(ul)    100x(ul)      Init Conc     Final Conc
H2O                           10         1000         --            --
2x Phusion Flash PCR MM       25         2500         2x            1x
AmpF6.4Sol                    2.5         250         10uM          .5uM
AmpR6.3.Indx                  2.5   (add separately)  10uM          .5uM
Circularized DNA Template     10    (add separately)  --            --
                              50
File:Cw 20151023 PhuPCR barcodes.pdf
  • Thermocycler Profile: (need to change to 98C denaturing)
98C 30sec
(98C 10sec -> 58C 30sec -> 72C 20sec)x8
(98C 10sec -> 72C 20sec)x12 cycles
72C 3min
Hold 15C