Sequencing (PE read)

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 Problem list:

  • how much free space on drive D is required for the run (maybe a table: type of run / disk space)
  • which volume of water is spent during PEM wash


[править] Preliminary

  • check the disk space (should be ### on drive D);
  • Kits required: Paired-End Cluster Generation Kit v4: box 2 of 2 (-20°C);
  • write (i) date and (ii) Flow Cell number on the box
  • thaw reagents in a beaker filled with room temperature water:

NB! For RMX, LMX2, BMX and AMX2: when only faint ice pieces are noticeable, place solutions on ice
  From Cluster Generation Kit box 2 of 2 (-20°C):

RMX    (Resynthesis Mix)
LMX2     (Linearization 2 Mix)
BMX     (Blocking Mix)
AMX2     (Amplification Mix 2)
APM2     (AMX2 Premix)
AT2     (Formamide)
HP2     (Seq Primer Mix2)
HP3     (0.1 N NaOH)
HT2     (Wash Buffer)

  • APM2, AT2, HP2, HP3, HT2 tubes:
» invert several times to mix
» centrifuge at 1krpm at room temperature for 1min
» leave on room tempearture

  • RMX, LMX2, BMX and AMX2 tubes:
» invert several times to mix
» pulse spin
» put on ice

[править] Prepare reagents

  1. label the 15mL tubes from the kit:

  2. 10RMX Resynthesis Mix
    11LMX2 Linearization 2 Mix
    12BMX Blocking Mix
    13AMX2 Amplification Mix2
    14APM2 AMX2 Premix
    15AT2 Formamide
    16HP2 Seq Primer Mix2
    19HP3 0.1 N NaOH
    21HT2 Wash Buffer

  3. prepare:
    • 0.1N NaOH in tube #19:
    2N NaOH 150µL HP3
    H2O2,85mL PW1
    • vortex and spin down the tube
  4. weigh the reagents and record weights and lot numbers in the check list:

[править] Cluster regeneration

  1. wash the PEM:
    • load a known volume of water onto the PE module (pos. 10-16,19,21)
    • click "OK" to resume the recipe and then "OK" to start the wash
    • click "OK" when water prime is complete and check remaining volume: should be ###
  2. load reagents on the PE module (place water tubes in rack) ⇒ “OK”
  3. NB! cluster regeneration takes ~4 hours, meanwhile prepare the sequencing reagents (see «Preparation of sequencing reagents» protocol)
  4. check volumes of reagents after reaction:

    positionIllumina Namesolutioninitial volumeshould remainused
    10RMXResynthesis Mix2ml0.65ml1.35ml
    11LMX2Linearization Mix 22ml0.65ml1.35ml
    12BMXBlocking Mix2ml0.1ml1.9ml
    13AMX2Amplification Mix 29ml3.93ml5ml
    14APM2AMX2 Premix6ml0.7ml5.3ml
    16HP2Sequencing Primer Mix 21.5ml0.15ml1.35ml
    19HP3Diluted (0.1 N NaOH)3ml1.1ml2.9ml
    21HT2Wash Buffer10ml2.8ml7.2ml

    [править] Sequencing: second read

    1. exchange the sequencing reagent tubes used for read 1 with fresh ones in order: 1, 3, 4, 5, 7, 6;
    2. click OK to resume the «GA2-PEM_2x36_PE_v8.3» recipe;
      first base incorporation begins
    3. remove reagent tubes from PE module, positions 9-21:
      • remove reagent tube;
      • wash tubing from washing bottle;
      • reinstall 15ml tube with ~10ml of mQ from the pre-run wash step;
    4. when 1st base incorporation is complete, click Cancel to perform refocusing:
      • open «Manual Control/Setup» window;
      • pump: [to: flowcell • solution: 3 • volume: 100 • asp. rate: 250 • disp. rate: 2500] ⇒ Enter;
      • perform refocusing (only Z, do not change XY settings, Focusing, p.8-9: setting Z roughly & accurately) and recalibrate the autofocus laser;
    5. click OK to resume the «GA2-PEM_2x36_PE_v8.3» recipe;
      imaging begins
    6. evaluate the first base report data:
      * number of clusters should be 90-120x103 per tile;
    7. * FC tilt: difference between min and max focus stage values should be less than 15000 nm;
      *intensity values:
      ConfidenceA & CGT
      High> 650> 1000> 1200
      Reasonable> 350> 650> 700
      Low< 250< 350< 400
    8. click OK to process sequencing.
      51 cycles run takes ~2,5days
    9. weigh the reagent bottles from PEM and record results;
    10. after the run
      • perform the post-run wash
      • weigh reagent bottles and record results

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