Chris:LabNotes/FateMapping/Calendar/2015/2015-3-13

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Target Capture - Using C1 amplicons and extended MDA from 2015_02_26 run w/ 9/22 probes (Part II)[edit]

Background[edit]

---------------------------Extended MDA Hemo KlenTaq (Strip A)------------------------------------
    rxn     Target (ng)         Probe:Target     Probe (ng)        Description
    1A      50ng (MDA)          500:1            3.69 (CW 9/22)    CW 9/22 gDNA MDA Control (Hemo KlenTaq)
    2A      50ng (gDNA)         500:1            3.69 (CW 9/22)    CW 9/22 gDNA Control (Hemo KlenTaq)
    3A      50ng (Extended G1)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well G1 (Hemo KlenTaq)
    4A      50ng (Extended H1)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well H1 (Hemo KlenTaq)
    5A      50ng (Extended A2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well A2 (Hemo KlenTaq)
    6A      50ng (Extended B2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well B2 (Hemo KlenTaq)
    7A      50ng (Extended C2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well C2 (Hemo KlenTaq)
    8A      0ng  (Extended NTC) --               3.69 (CW 9/22)    CW 9/22 Extended NTC (Hemo KlenTaq) <- NTC from the extension MDA reaction
--------------------------------Extended MDA Phusion (Strip B)------------------------------------
    rxn     Target (ng)         Probe:Target     Probe (ng)        Description
    1B      50ng (MDA)          500:1            3.69 (CW 9/22)    CW 9/22 gDNA MDA Control (Phusion)
    2B      50ng (gDNA)         500:1            3.69 (CW 9/22)    CW 9/22 gDNA Control (Phusion)
    3B      50ng (Extended G1)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well G1 (Phusion)
    4B      50ng (Extended H1)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well H1 (Phusion)
    5B      50ng (Extended A2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well A2 (Phusion)
    6B      50ng (Extended B2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well B2 (Phusion)
    7B      50ng (Extended C2)  500:1            3.69 (CW 9/22)    CW 9/22 Extended C1 Well C2 (Phusion)
    8B      0ng  (Extended NTC) --               3.69 (CW 9/22)    CW 9/22 Extended NTC (Phusion)
---------------------------------C1 Hemo KlenTaq (Strip C)----------------------------------------
    rxn     Target (ng)         Probe:Target     Probe (ng)        Description
    1C      50ng (MDA)          500:1            3.69 (CW 9/22)    CW 9/22 gDNA MDA Control (Hemo KlenTaq)
    2C      50ng (gDNA)         500:1            3.69 (CW 9/22)    CW 9/22 gDNA Control (Hemo KlenTaq)
    3C      50ng (C1 Well G1)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well G1 (Hemo KlenTaq)
    4C      50ng (C1 Well H1)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well H1 (Hemo KlenTaq)
    5C      50ng (C1 Well A2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well A2 (Hemo KlenTaq)
    6C      50ng (C1 Well B2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well B2 (Hemo KlenTaq)
    7C      50ng (C1 Well C2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well C2 (Hemo KlenTaq)
    8C      0ng  (NTC)          --               3.69 (CW 9/22)    CW 9/22 NTC (Hemo KlenTaq) <- This is just water added into the reaction
------------------------------------C1 Phusion (Strip D)------------------------------------------
    rxn     Target (ng)        Probe:Target     Probe (ng)        Description
    1D      50ng (MDA)          500:1            3.69 (CW 9/22)    CW 9/22 gDNA MDA Control (Phusion)
    2D      50ng (gDNA)         500:1            3.69 (CW 9/22)    CW 9/22 gDNA Control (Phusion)
    3D      50ng (C1 Well G1)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well G1 (Phusion)
    4D      50ng (C1 Well H1)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well H1 (Phusion)
    5D      50ng (C1 Well A2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well A2 (Phusion)
    6D      50ng (C1 Well B2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well B2 (Phusion)
    7D      50ng (C1 Well C2)   500:1            3.69 (CW 9/22)    CW 9/22 C1 Well C2 (Phusion)
    8D      0ng  (NTC)          --               3.69 (CW 9/22)    CW 9/22 NTC (Phusion)

Procedure[edit]

Prep KLN mix solution (Strips A and C)[edit]

  • We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued)
  • First, perform the following dilutions:
    • Dilute dNTP 1/10d (2ul dNTP + 18ul H2O) (10mM/nt -> 1mM/nt)
  • Mastermix recipe
Component            Stock conc     Final conc     1x(ul)     MM Vol (18x) (ul)     
Hemo KlenTaq         100%           20%            0.4        7.2            
AmpLigase            5U/ul          0.5U/ul        0.2        3.6            
dNTP                 1mM            100uM          0.2        3.6     
10x AmpLigase Buff   10x            1x             0.2        3.6            
H2O                                                1.0        18           
  • Aliquot 4.5ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions
  • Add 2ul KLN mix to Strips A and C rxns 1->8 using multichannel

Prep Phusion/AmpLigase mix solution (Strips B and D)[edit]

  • We are using the Phusion/AmpLigase mix described in the paper
  • First, perform the following dilutions:
    • Dilute dNTP 1/10d (2ul dNTP + 18ul H2O) = use same diluted dNTP as above
    • Dilute NAD+ 1/10d (15ul NAD+ + 135ul H2O)(50mM -> 5mM)
  • Mastermix recipe
Component              Stock conc     Final Amount     1x(ul)              MM Vol (18x) (ul)
NAD+                   5mM            40nmol           8.0 (after dil)     144
dNTP                   1mM            600pmol          0.6 (after dil)     10.8
Betaine                5M             15umol           3.0                 54
10x AmpLigase Buff     10x            1x               2.0                 36
AmpLigase              5U/ul          10U              2.0                 36
Phusion HF DNA Pol     2000U/ml       6.4U             3.2                 57.6
Water                                                  1.2                 21.6
  • Aliquot 45ul Phusion/AmpLigase mix to each well of a strip tube so can use multichannel when adding to the reactions
  • Add 20ul Phusion/AmpLigase mix to Strips B and D rxns 1->8 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 (34x) (ul)
ExoI          20U/ul         20U              1           34
ExoIII        200U/ul        100U             0.5         17
Water                                         0.5         17
  • Aliquot 8.5ul 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
    • Because Hemo KlenTaq and Phusion have different reaction volumes (24ul vs 42ul), we'll need to set up two different PCR reactions for each. The reason why is because we want to keep the input amount of DNA approximately the same (i.e. input half of the volume of the reactions for PCR)
  • Note: Since we are not going to do qPCR, we don't need to add SYBR into rxn mix
  • Mastermix recipe (Strip A, 24ul reaction from capture)
Component                     1x(ul)     16.5x(ul)    Init Conc     Final Conc
H2O                           28          462         --            --
2x Phusion Flash PCR MM       50          825         2x            1x
AmpF6.4Sol                     5         82.5         10uM          .5uM
AmpR6.3.Indx                   5    (add separately)  10uM          .5uM
Circularized DNA Template     12    (add separately)  --            --
                              100
  • Mastermix recipe (Strip B, 42ul reaction from capture)
Component                     1x(ul)     16.5x(ul)    Init Conc     Final Conc
H2O                           19         313.5        --            --
2x Phusion Flash PCR MM       50          825         2x            1x
AmpF6.4Sol                     5          82.5        10uM          .5uM
AmpR6.3.Indx                   5    (add separately)  10uM          .5uM
Circularized DNA Template     21    (add separately)  --            --
                              100
Strip A
rxn          Indx     Indx Seq (RC)
1            20       CCTGTC
2            21       CAACCC
3            22       CTCGGA       
4            23       TCGAAA
5            24       ATTCGC
6            25       TACTGC
7            26       TCGTGA
8            19       GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture
-------------------
Strip B
rxn          Indx     Indx Seq (RC)
1            27       TACGCG
2            28       AGGTGC
3            29       ACGAAC
4            30       TTAGTG
5            31       CACCAC
6            33       CGCAAC <- Skip Index 32 because was identified as bad and not sure if they changed the sequence for these primers yet
7            34       AACTGA
8            19       GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture
-------------------
Strip C
rxn          Indx     Indx Seq (RC)
1            35       ATCGGG
2            36       CAAGCA
3            37       GCTACA
4            38       TCACGC
5            39       CCAGAG
6            40       TGACGG
7            41       GGGAAC
8            19       GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture
-------------------
Strip D
rxn          Indx     Indx Seq (RC)
1            42       GAAAAG
2            43       AGTGCC
3            44       GCATCC
4            45       ACTACG
5            46       CATTTC
6            47       CTCTCC
7            48       ACGTTA
8            19       GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture
  • 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

Results[edit]

  • Below is the gel image for Strip A (Low Mass Ladder, x, rxn's 1->8)

File:Cw ZhangLab 2 2015-03-13 16hr 48min pp cap pcr A.jpg

  • Below is the gel image for Strip B (Low Mass Ladder, rxn's 1->8)

File:Cw ZhangLab 2 2015-03-13 16hr 46min pp cap pcr B.jpg

  • Below is the gel image for Strip C (Low Mass Ladder, rxn's 1->8)

File:Cw ZhangLab 2 2015-03-13 16hr 51min pp cap pcr C.jpg

  • Below is the gel image for Strip D (Low Mass Ladder, rxn's 1->8)

File:Cw ZhangLab 2 2015-03-13 16hr 49min pp cap pcr D.jpg