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, P44G 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 (columsn 1->6) using multichannel

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

  • 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

  • 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 volume into each PCR reaction)
Component                     1x(ul)    100x(ul)      Init Conc     Final Conc     Make two of these in order for total of 52 rxns
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
  • 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 enzymes (when combining the different libraries, make sure combine in equal amounts based on gel quantification; sequence the two enzyme conditions in two separate lanes in HiSeq RapidRun). However, we want to make sure that the indices for each individual single cell amplicon is different
    • The following file contains information on the pooling and indices used for the different reactions: File:Cw 20150812 PhuPCR index-seqs.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

Results

  • Below are the gel images from the capture done in this experiment:

File:Cw ZhangLab 2 2015-08-13 10hr 48min PPCap Gel 1A-H,2A-D.jpg

File:Cw ZhangLab 2 2015-08-13 10hr 50min PPCap Gel 2E-H, 3A-H.jpg

File:Cw ZhangLab 2 2015-08-13 10hr 52min PPCap Gel 4A-H,5A-D.jpg (Note that one of the lanes showed nothing because loading volume was very low...probably pipetting error)

File:Cw ZhangLab 2 2015-08-13 10hr 54min PPCap Gel 5E-H,6A-H.jpg

File:Cw ZhangLab 2 2015-08-13 12hr 01min PPCap Gel 7A-H,8A-D.jpg

File:Cw ZhangLab 2 2015-08-13 12hr 02min PPCap Gel 8E-H,9A-H.jpg

File:Cw ZhangLab 2 2015-08-13 12hr 04min PPCap Gel 10A-H,11A-D.jpg

File:Cw ZhangLab 2 2015-08-13 12hr 05min PPCap Gel 11E-H,12A-H.jpg

  • Note that while pooling and size selecting, I needed to do Qiaquick purification in order for the gel to look okay (otherwise the bands are not clear due to the high amounts of salt). Otherwise, the gels looked fine as shown below:

File:Cw ZhangLab 2 2015-08-13 17hr 13min PPCap pre-size selection L,pool1-4.jpg

File:Cw ZhangLab 2 2015-08-13 17hr 15min PPCap pre-size selection pool5,L,pool6-8.jpg

File:Cw ZhangLab 2 2015-08-13 17hr 30min PPCap post-size selection L,pool1-4.jpg

File:Cw ZhangLab 2 2015-08-13 17hr 31min PPCap post-size selection pool5,L,pool6-8.jpg