Chris:LabNotes/FateMapping/Calendar/2015/2015-8-12

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Target Capture - Using C1 amplicons and extended MDA from 2015_08_10 C1 run (WZTO BA9, R55E BA9) w/ 9/22/2014 probes (Hemo and Phusion Reactions) (Part II)

Background

Procedure

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

  • 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 using multichannel

Prep Phusion mix solution (6 strips, 8 rxn's each + 2 NTC)

  • 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 <- We were short 40ul so had to add 40ul of water more in order to make up for the volume difference
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 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

  • We want to then add 20U ExoI and 100U ExoIII
  • Mastermix recipe
Component     Stock Conc     Final Amount     1x(ul)      MM Vol (108x) (ul)
ExoI          20U/ul         20U              1           108
ExoIII        200U/ul        100U             0.5         54
Water                                         0.5         54
  • Aliquot 26ul 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

  • 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 concentration into each PCR reaction)
Component                     1x(ul)     26x(ul)      Init Conc     Final Conc     Make two of these in order for total of 52 rxns
H2O                           14          364         --            --
2x Phusion Flash PCR MM       25          650         2x            1x
AmpF6.4Sol                    2.5          65         10uM          .5uM
AmpR6.3.Indx                  2.5   (add separately)  10uM          .5uM
Circularized DNA Template      6    (add separately)  --            --
                              50
  • Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>)
    • Note: We are overlaping indices for single cells across the two different probe sets (when combining the different libraries, make sure combine in equal amounts based on gel quantification). However, we want to make sure that the indices for the Hemo/Phusion probe capture reactions are different in order to validate that there is a difference between the two enzyme captures
---------------------------9/22/2014 Probes (Strip A)------------------------------------
    rxn     Index     Indx Seq (RC)
    1A      1         ATCACG
    2A      2         CGATGT
    3A      4         TGACCA <- Indx 3 is bad
    4A      5         ACAGTG
    5A      6         GCCAAT
    6A      7         CAGATC
    7A      8         ACTTGA
    8A      10        TAGCTT <- Index 9 is bad
---------------------------9/22/2014 Probes (Strip B)------------------------------------
    rxn     Index     Indx Seq (RC)
    1B      11        GGCTAC
    2B      12        CTTGTA
    3B      13        CCATGA
    4B      14        AAGACA
    5B      15        CTTCCT
    6B      16        GGGGTT
    7B      17        CTCATC
    8B      18        AGTTCA
---------------------------9/22/2014 Probes (Strip C)------------------------------------
    rxn     Index     Indx Seq (RC)
    1C      19        GACGCA
    2C      20        CCTGTC
    3C      21        CAACCC
    4C      22        CTCGGA
    5C      23        TCGAAA
    6C      24        ATTCGC
    7C      25        TACTGC
    8C      26        TCGTGA

    NTC     3         TTAGGC <- Indx 3 is bad, so can use for NTC
---------------------------5/23/2015 Probes (Strip A)------------------------------------
    rxn     Index     Indx Seq (RC)
    1A      1         ATCACG
    2A      2         CGATGT
    3A      4         TGACCA <- Indx 3 is bad
    4A      5         ACAGTG
    5A      6         GCCAAT
    6A      7         CAGATC
    7A      8         ACTTGA
    8A      10        TAGCTT <- Index 9 is bad
---------------------------5/23/2015 Probes (Strip B)------------------------------------
    rxn     Index     Indx Seq (RC)
    1B      11        GGCTAC
    2B      12        CTTGTA
    3B      13        CCATGA
    4B      14        AAGACA
    5B      15        CTTCCT
    6B      16        GGGGTT
    7B      17        CTCATC
    8B      18        AGTTCA
---------------------------5/23/2015 Probes (Strip C)------------------------------------
    rxn     Index     Indx Seq (RC)
    1C      19        GACGCA
    2C      20        CCTGTC
    3C      21        CAACCC
    4C      22        CTCGGA
    5C      23        TCGAAA
    6C      24        ATTCGC
    7C      25        TACTGC
    8C      26        TCGTGA

    NTC     3         TTAGGC <- Indx 3 is bad, so can use for NTC
  • 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