FlowCell processing

Материал из Zbio

Перейти к: навигация, поиск
NB! post-PCR operation

Содержание


[править] Preliminary

    • if necessary, book CS
    * Kits required: Single-Read Cluster Generation Kit v4: box 1 of 1 (-20°C); Kits required: Paired-End Cluster Generation Kit v4: box 1 of 2 (-20°C);
    • thaw reagents in a beaker filled with room temperature water:
    NB! For BMX and LS1: when only faint ice pieces are noticeable, place solutions on ice NB! For BMX and LMX1: when only faint ice pieces are noticeable, place solutions on ice
      From Cluster Generation Kit −20°C box:
    BMX    (Blocking Mix)
    ::LS1     (linearisation mix)  ::LMX1     (linearisation mix 1)
    HP3     (2N NaOH)
    ::HP4     (Sequencing primer buffer) ::genomic DNA sequencing primer :: HP1 (Sequencing primer mix 1)
    * HP3, HP4 and seq. primer tubes: *HP3and HP1 tubes:
    " invert several times to mix
    " centrifuge at 1krpm at room temperature
    " leave on room tempearture

    * BMX and LS1 tubes: * BMX and LMX1 tubes:
    " invert several times to mix
    " pulse spin
    " put on ice

    HT1    (Hybridization Buffer)
    HT2     (Wash buffer)
    PW1     (water from the Sequencing kit v4)

    * Check, that you have 3-amino-1-propanol (3-APL)

    [править] Prepare reagents

    NB! store all components on ice
    1. label tubes and store them on ice:
      • tubes from the kit:
      #4     BMX (Blocking Mix)
      #3    HT2 (Wash Buffer)
      ::#7     HP4 (Sequencing primer buffer) ::#7     HP1 (Sequencing primer mix 1) ::#15    LS1 (linearisation mix) ::#14    LMX1 (linearisation mix 1)
      #12    HT1 (hybridization buffer)
      • 1.7ml screw-cap tubes:
      #17     0.1N NaOH
    2. 0.1N NaoH in tube #17:
      HP3 (2N NaOH)75µl
      PW1 (water)2x712.5µl
      • mix by inverting
      • pulse spin
    3. linearization mix in tube #15: * add 1.4µl of 3-amino-1-propanol (3-APL) to the LS1 (linearisation mix) * mix by inverting * pulse spin
    4. sequencing primer mix in tube #7: * add 6.6µl of genomic DNA sequencing primer to the HP4 (Sequencing primer buffer) * mix by inverting * pulse spin
    5. record the weights/ volumes of the reagents in the check-list
    6. take following things to CS:
      • flowcell with clusters;
      • amplification manifold (from the cluster generation step);
      • storage buffer (for the case of problems with manifold);
      • 1ml and 200µl pipetts;




    [править] Linearization, blocking and primer hybridization

    1. if necessary: turn on CS, wait 30 s, turn on computer, empty the waste container;
    2. wipe the table and the CS with a wet paper towel;
    3. wash CS, ~10min: * change water in tubes: 3,4,7,12,15,17; * change water in tubes: 3,4,7,12,14,17; * open «SR_Linearization_Blocking_Primerhyb_v7» recipe; * open «PE_2P_R1Prep_Linearization_CombinedBlocking_LONG_PrimerHyb_v7 [1]» * select chemistry «WashLinearizationBlockingPrimerHybLines», «Start», «OK», * select chemistry «WashREAD1LinerizationandCombinedblockingprimerhybLines», «Start», «OK»,
      «User Wait» after the washing step and follow messages on the screen

    4. load reagents: * remove H2O from tubes in reagent positions 3,4,7,12,15,17 ⇒ OK * remove H2O from tubes in reagent positions 3,4,7,12,14,17 ⇒ OK
      prime air 3,4,7,12,15,17;
      * load reagents in positions 3,4,7,12,15,17 ⇒ Cancel; * load reagents in positions 3,4,7,12,14,17 ⇒ Cancel;
    5. load FC:
      • remove liquid from the washing bridge [26 · 40µl/min · 120µl];
      • remove washing bridge;
      • install Flow Cell and amplification manifold;
      * manually prime line 12 (HT1 hybridization buffer); * pump HT1 hybridization buffer and check the proper flow through all channels [12 · 60µl/min · 100µl]; * pump a small air gap [26 · 40µl/min · 10µl];
    6. program:
      • resume the recipe ⇒ OK
      <>* linearisation:</span>
      prime reagents 3,15
      20°C · 1°C/s
      [15 · 15µl/min · 150µl]
      [3 · 60µl/min · 95µl]
      <> <> * Flow check
      prime reagents 3,14,12
      pump reagents 12 ⇒ OK
      [12•30µl7min•80]
      check for proper flow in all 8 lanes
       — flow is regular ⇒ OK
      −0 flow is irregular ⇒ Cancel, repeat the step
      <> * READ1 Linearization
      20°C•1°C/s
      [3•60µl/min•95µl]
      [14•60µl/min•95µl]
      37,9°C•1°C/s
      wait 30 min
      20°C•1°C/s
      [3•60µl/min•95µl]

      <> * blocking: :: prime reagents 12,4 :: prime reagent 4
      38°C · 1°C/s
      [4 · 60µl/min · 95µl]
      9x cyclic pumping: [4 · 15µl/min · 5µl]
      wait 3 min
      60°C · 1°C/s
      [4 · 60µl/min · 25µl]
      wait 15 min
      20°C · 1°C/s
      [3 · 60µl/min · 95µl]
      [12 · 60µl/min · 95µl]

      <> * primer hybridization:
      prime reagents 7,17
      20°C · 1°C/s
      [17 · 60µl/min · 95µl]
      [3 · 60µl/min · 95µl]
      [7 · 60µl/min · 95µl]
      60°C · 1°C/s
      wait 5 min
      40°C · 1°C/s
      [3 · 60µl/min · 95µl]
      20°C · 1°C/s
      [12 · 60µl/min · 95µl]
      «User Wait»

    7. remove FC:
      • air gap [26 · 30µl/min · 40µl];
      • unclip «reagent delivery port» and «central clamp» of amplification manifold;
      • remove FC and accurately clean it with water and lens cleaning tissue;
      • transfer FC to the Genome Analyser;
      • install empty FC and attach the amplification manifold back.

    8. CS post-wash:
      • wash amplification manifold (see «Washing of manifolds»);
      • install washing bridge;
      * pour fresh water in water-tubes of the used positions X = 3,4,7,12,15,17: * pour fresh water in water-tubes of the used positions X = 3,4,7,12,14,17:
      o remove tube with reagent;
      o air gap [X · 240µl/min · 30µl], during the air pumping rinse the end of the tubing using washing bottle;
      o attach tube with water;
      * open «SR_Linearization_Blocking_Primerhyb_v7» recipe; * open «PE_ZP_R1Prep_Linearization_Combined Blocking_LONG_Primer Hyb_v7» recipe. * select chemistry «WashLinerizationBlockingPrimerHybLines», «Start», «OK»,~10 min * select chemistry «WashRead1LinerizationandCombinedblockingprimerhybLines», «Start», «OK»,~10min
    9. close the program; turn off computer, turn off CS. </ol>

      [править] Notes

      1. we have changed the time of primer hybridization from 5 min in the Illumina recipe to 15 min, fluorescent signal after first base incorporation seem to be better then

      * If only linearization and blocking are performed, prepare reagents for positions: 3,4,12,15 and stop manually before primer hybridization. Another possibility is to fill positions 7 and 17 with HT1 hybridization buffer not to worry about missing the right time. * If only linearization and blocking are performed, prepare reagents for positions: 3,4,12,14 and stop manually before primer hybridization. Another possibility is to fill positions 7 and 17 with HT1 hybridization buffer not to worry about missing the right time.

      • Volumes of reagents after reaction:
      |#7||HP4 (Sequencing primer buffer)||1.3ml||0.35ml||1ml |#7||HP1 (Sequencing primer mix)||1.3ml||0.35ml||1ml |#15||LS1 (linearisation mix)||1.3ml||0.3ml||1ml |#14||LMX1 (linearisation mix)||1.3ml||0.25ml||1.1ml
      positionreagent initial volume should remain used
      #3HT2 (Wash Buffer)7ml3ml3.4ml
      #4BMX (Blocking Mix)2ml0.53ml1.7ml
      #12HT1 (hybridization buffer)13ml10ml3ml
      #170.1N NaOH1.5ml0.5ml1ml



Личные инструменты


Инструменты




molbiol.ru  ·  redactor@molbiol.ru  ·  реклама

 ·  Викимарт - все интернет-магазины в одном месте  ·  Доска объявлений Board.com.ua  · 
--- сервер арендован в компании Hetzner Online, Германия ---
--- администрирование сервера: Intervipnet ---


molbiol.ru - методы, информация и программы для молекулярных биологов     Rambler's Top100 Rambler