Multiple primer hyb.

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problems/questions
  • insert primers dilution calculation (from seq.molbiol.ru)


Содержание


NB! post-PCR operation
NB! This protocol is both for SR and PE (Read1) flow cells. Linearization and blocking should already be performed


[править] Preliminary

  • if necessary, book the Cluster Station
  • kits required :
» Single-Read Cluster Generation Kit v4: box 1 of 1 (-20°C); flowcell; hybr. and amp. manifolds
  • thaw reagents in a beaker filled with room temperature water:

  From Cluster Generation Kit -20°C box:

HP3     (2N NaOH)
HP4     (Sequencing primer buffer)
sequencing primers
» invert several times to mix
» centrifuge at 1krpm at room temperature
» leave on room tempearture
  • prepare reagents from the previuos Cluster Station steps:
HT1    (Hybridization Buffer), labeled #12
HT2     (Wash buffer), labeled #3
PW1     (water from the Sequencing kit v4)



[править] Prepare reagents

NB! store all components on ice
  1. label strips:
    # F NaOH, 0.1N
    # G HT2 (wash buffer)
    # H sequencing primers (mark each tube)
    # I HT2 (wash buffer)
    # J HT1 (hybridization buffer)
    # K empty
    # W H2O for washing manifold
  2. prepare primer mix in hybridization buffer (recommended dilution at least 1:200): insert calculation form
  3. fill the strips:
    # G 100µl of HT2 (wash buffer)
    # H 100µl of primer mixture
    # I 100µl of HT2 (wash buffer)
    # J 100µl of HT1 (hybridization buffer)
    # W 240µl of H2O
  4. dilute 2N NaOH (HP3) to 0.1N NaOH:
    • in 1,5ml tube: 779µl PW1 (water)+41µl HP3 (2N NaOH)
    • mix by inverting the tube
    • pulse spin
    • fill the tube strip # F with 100µl of 0.1N NaOH
  5. take following things to CS:
    • flowcell with clusters;
    • hybridization manifold (from the cluster generation step);
    • HT1 (hybridization buffer) (for the case of problems with manifold);
    • 1ml and 200µl pipettes;




[править] 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. open «SR_MultiplePrimerhyb_only_LONG PRIMER_v7» recipe;
  4. install flowcell:
    • remove liquid from the washing bridge [26 · 40µl/min · 120µl];
    • remove washing bridge;
    • install flowcell, tube strip holder and hybridization manifold;
  5. put the # J1 strip with HT1 (hybridization buffer) in the strip holder;
  6. pump HT1 (hybridization buffer) and check (IMPORTANT!) the liquid flow through the all channels [26 · 60µl/min · 100µl];
  7. remove the # J1 strip and pump a small air gap [26 · 40µl/min · 10µl];
    «User Wait» after the washing step
  8. follow messages on the screen:
    • install the FC and the hybr. manifold ⇒ OK;
    • strip # F ⇒ OK
    [26 · 60µl/min · 75µl]
    • strip # G ⇒ OK
    [26 · 60µl/min · 75µl]
    • strip # H ⇒ OK
    [26 · 60µl/min · 75µl]
    60°C · 1°C/s
    wait 15 min
    40°C · 1°C/s
    • strip # I ⇒ OK
    [26 · 60µl/min · 75µl]
    20°C · 1°C/s
    • strip # J2 ⇒ OK
    [26 · 60µl/min · 75µl]
  9. remove FC:
    • strip # O;
    • air gap [26 · 15µl/min · 40µl]
    • unclip «reagent delivery port» & «central clamp» of hybridization manifold;
    • remove FC and accurately clean it with water and lens cleaning tissue;
    • tranfer flowcell to the Genome Analyser;
    • install empty FC and attach the hybridization manifold back;
  10. wash hybridization manifold (see «Washing of manifolds»);
  11. close the program; turn off computer, turn off CS.




[править] Notes

  • Volumes and reagents after reaction (per tube):
positionreagent initial volume should remain
# FNaOH, 0.1N100µl25µl
# GHT2 (wash buffer)100µl)25µl
# Hsequencing primers100µl25µl
# IHT2 (wash buffer)100µl25µl
#JHT1 (hybridization buffer)100µl25µl

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