HS read 2 run

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[править] Preparing ICR for Read 2


[править] Thaw Reagents for 101cycles

  1. [  ]  leave LRP (Long Read DNA Polymerase) at -20°C until mixing to ICR
  2. [  ]  replace two tubes of LFN (nucleotides) from -20°C to 4°C and thaw overnight
  3. [  ]  invert each tube several times and ensure there are completely thawed.
  4. [  ]  use ICR directly from 4°C



[править] Prepare ICR for 101 cycles

  1. [  ]  add the contents of two tubes of LFN to the bottle containing 47 ml of ICR.
  2. [  ]  rinse each tube of LFN with ICR to ensure that all LFN is transferred
  3. [  ]  aliquot 1.1 ml of LRP into the ICR/LFN mix
  4. [  ]  cap the bottle and invert several times to mix
  5. [  ]  set the mix on ice until loading reagents onto the HiSeq.



[править] Preparing PE- Reagents

»the following reagents are used for Read 2 (resynthesis) and are provided in HiSeq Read 2 Cluster Resynthesis Kit, which is box 2 of the HiSeq Paired-End Cluster Generation Kit.
»take following reagents from - 20°C storage and thaw them at RT or in a beaker with RT water:
• HT2 (Wash Buffer)
• HP3 (2 N NaOH)
• AT2 (100% Formamide)
• APM2 (AMX2 Premix)
• AMX2 (Amplification Mix 2)
• BMX (Blocking Mix)
• LMX2 (Linearization Mix 2)
• RMX (Resynthesis Mix)
• HP2 (Sequencing Primer Mix 2) (for standard paired-end runs)

[править] Prepare Reagents

Prepare RMX, LMX2, BMX, and AMX2 :

  1. [  ]  invert each thawed tube to mix the reagent
  2. [  ]  put reagents on ice until ready for loading onto the HiSeq.

Prepare APM2, AT2, and HT2 :

  1. [  ]  invert each thawed tube to mix the reagent.
  2. [  ]  keep reagents at room temperature until loading onto the HiSeq.

Prepare HP3 Dilute HP3 (2 N NaOH) to 0.1 N NaOH prior to loading it onto the HiSeq.

  1. [  ]  vortex the thawed tube of HP3 and then pulse spin down.
  2. [  ]  transfer 2.85 ml of PW1 (provided in the HiSeq Sequencing Kit) into a 15 ml falcon tube and add 150 μl of HP3
  3. [  ]  vortex and spin down
  4. [  ]  keep the NaOH mix on RT until loading onto the HiSeq

Prepare HP2 (Standard PE runs)

  1. [  ]  vortex both thawed tubes and spin down
  2. [  ]  keep the primermix on RT until loading onto the HiSeq



[править] Loading ICR for Read 2 onto the HiSeq

» before starting Read 2 load fresh ICR

  1. [  ]  record the weight of ICR on the lab tracking form
  2. [  ]  open the reagent door, raise the sippers for the reagent rack and replace (position 1) the fresh ICR with existing mix
  3. [  ]  change the funnel cap to the fresh bottle of ICR mix
  4. [  ]  lower the sipper for the reagent rack into the reagent bottles and make sure they are centered inside the bottles



[править] Loading of PE Reagents onto the HiSeq

» load the PE-reagents onto the PE -reagent rack before starting of read 2-resynthesis

  1. [  ]  record the weight of each reagent on the lab tracking form
  2. [  ]  make sure the PE rack is not in use for Read 2 resynthesis or Index Read preparation on the other flow cell.
  3. [  ]  raise the sippers for the PE reagent rack, remove the rack and put in the PE-reagent tubes
    Reagent description:
    10 RMX Resynthesis Mix
    11 LMX2 Linearization Mix 2
    12 BMX Blocking Mix
    13 AMX2 Amplification Mix 2
    14 APM2 AMX2 Premix
    15 AT2 100% Formamide
    16 HP7 Sequencing Primer Mix 2
    18 HP3 Denaturation Solution
    19 HT2 Wash Buffer
  4. [  ]  lower the sippers into the paired-end reagent tubes and make sure that the sippers are centred inside the tubes
  5. [  ]  close the reagent door and press next to resume the run





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