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(Created page with "=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== *T...")
 
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==Background==
==Background==
*This is a continuation of the experiment started on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11>
*This is a continuation of the experiment started on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11>
*As a reminder, the reactions of rthis experiment are described below  (46 C1 amplicons in total + 2NTC, all with 9/22/14 probes; one set with Hemo and another with Phusion)
*As a reminder, the reactions for this experiment are described below  (46 C1 amplicons in total + 2NTC, all with 9/22/14 probes; one set with Hemo and another with Phusion)
==Procedure==
==Procedure==
===Prep KLN mix solution (6 strips, 8 rxn's each)===
===Prep KLN mix solution (6 strips, 8 rxn's each)===
Line 16: Line 16:
  H2O                                                1.0        54
  H2O                                                1.0        54
*Aliquot 13ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions
*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
*Add 2ul KLN mix to all strips (columsn 1->6) using multichannel


===Prep Phusion mix solution (6 strips, 8 rxn's each + 2 NTC)===
===Prep Phusion mix solution (6 strips, 8 rxn's each)===
*First, perform the following dilutions:
*First, perform the following dilutions:
**Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) (same as above)
**Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) (same as above)
Line 29: Line 29:
  10x AmpLigase Buff    10x            1x              2.0                106
  10x AmpLigase Buff    10x            1x              2.0                106
  AmpLigase              5U/ul          10U              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'''
  Phusion HF DNA Pol    2000U/ml      6.4U            3.2                169.6
  Water                                                  1.2                63.6
  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
*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
*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
*Thermocycler (continued): Add KLN or Phusion mix to each rxn -> '''Incubate 60C 4hr''' -> 94C 2min
Line 50: Line 50:
*We are going to use the target captured circularized DNA as template for Phusion reaction
*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'''
*'''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)
*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)     26x(ul)      Init Conc    Final Conc    '''Make two of these in order for total of 52 rxns'''
  Component                    1x(ul)   100x(ul)      Init Conc    Final Conc    '''Make two of these in order for total of 52 rxns'''
  H2O                          14          364         --            --
  H2O                          10        1000         --            --
  2x Phusion Flash PCR MM      25         650         2x            1x
  2x Phusion Flash PCR MM      25         2500         2x            1x
  AmpF6.4Sol                    2.5         65         10uM          .5uM
  AmpF6.4Sol                    2.5         250         10uM          .5uM
  AmpR6.3.Indx                  2.5  (add separately)  10uM          .5uM
  AmpR6.3.Indx                  2.5  (add separately)  10uM          .5uM
  Circularized DNA Template     6   (add separately)  --            --
  Circularized DNA Template     10   (add separately)  --            --
                               50
                               50
*Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>)
*Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>)
**<span style="color:red">'''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'''</span>
**<span style="color:red">'''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'''</span>
---------------------------'''9/22/2014''' Probes (Strip A)------------------------------------
**The following file contains information on the pooling and indices used for the different reactions: [[File:cw_20150812_PhuPCR_index-seqs.pdf]]
    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)
*Thermocycler Profile: (need to change to 98C denaturing)
  98C 30sec
  98C 30sec

Revision as of 23:21, 14 August 2015

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 (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