Cluster generation

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problems/questions
  • insert the option "number of libraries" in the dilution recipe calculation"


NB! post-PCR operation

Содержание


[править] Preliminary

  • if necessary, book CS
  • kits required :

  Single read:

» Single-Read Cluster Generation Kit v4: box 1 of 1 (-20°C); flowcell; hybr. and amp. manifolds
» Sequencing kit TruSeq SBS v5: PW1 (water)

  Paired-End:

» Paired-End Cluster Generation Kit v4: box 1 of 2 (-20°C); flowcell; hybr. and amp. manifolds
» Sequencing kit TruSeq SBS v5: PW1 (water)
  • determine loading concentration for libraries
  • (if not already done)thaw 10-20nM library solutions by finger tapping and place on ice
  • calculate dilution recipe for each library:

opt.conc.No. of channels    volume
denat. libraryhybridization buffer

  • thaw reagents in a beaker filled with room temperature water:

NB! For AMX1 and HFE: when only faint ice pieces are noticeable, place solutions on ice

  From Cluster Generation Kit -20°C box:

AMX1    (Amplification Mix)
HT2     (Wash Buffer);
AT1     (100% Formamide);
APM1    (AMX1 Premix)
HT1     (Hybridization Buffer)
HFE     (1xPhusion Master Mix, Finnzymes Oy)
HP3     (2N NaOH)

  from Sequencing Kit:

PW1     (Water)
  • APM1, HT2, AT1, HT1, HP3, PW1 tubes:
» invert several times to mix
» centrifuge at 1krpm at room temperature
» leave at room tempearture
  • AMX1 tube:
» invert several times to mix
» shake down to remove drops from the cap
» put on ice
  • HFE tube:
» invert several times to mix
» pulse spin
» put on ice



[править] Prepare reagents

NB! store all components on ice, except for the strip #B with template (room temperature)

  1. [  ]  label:
    • 8-strips:
    #A    hybridization buffer
    #B    template (write the number of library on each tube)
    #C    wash buffer
    #D    amplif. premix
    #E    extension mix
    #F    0.1N NaOH
    #G    wash buffer
    #0    empty

    • 1.7ml tube:
    0.1N NaOH   

    • 50ml tubes ready to use from the kit:
    #1     AMX1 (Amplification Mix)
    #3    HT2 (Wash Buffer)
    #9     AT1 (100% Formamide)
    #11    APM1 (AMX1 Premix)
    #12    HT1 (hybridization buffer)

  2. [  ]  fill the strips:
    #A    8 x 140µl HT1 (hybridization buffer)
    #C    8 x 100µl HT2 (wash buffer)
    #D    8 x 100µl APM1 (ampl.premix)
    #E    8 x 120µl HFE (extention mix)
    #G    8 x 100µl HT2 (wash buffer)

  3. [  ]  0.1 N NaOH :
    • in 1,7ml 0.1N NaOH tube: 950µl PW1 (water)+50µl HP3 (2N NaOH)
    • transfer 8 x 100µl in strip #F (0.1N NaOH)

  4. [  ]  denature library:
    conc.stock
    library1nM
    NaOH0.1N2N
    EBQiagen  

    • vortex, spin down, 5min RT
    • prepare tubes with hybridization buffer (one tube per library) and transfer calculated volume (at least 2µl) of denatured library in each
    • vortex, spin down, transfer 120µl of template per channel #B (template)
    • keep strip #B at RT till loading on the FlowCell


  5. [  ]  take following things to CS:
    • hybridization manifold
    • amplification manifold
    • hybridisation buffer (for the case of problems with manifold)
    • 1ml and 200µl pipettes



[править] Cluster generation

  1. turn on CS, wait 30 s, turn on computer;
  2. empty the waste container, wipe the CS and table with a wet paper towel;
  3. wash CS, ~10min:
    • change water in tubes: 1,3,9,11,12
    • open "SR_Amplification_only_v7" or "PE_Amplification_only_v7" recipe
    • select chemistry "WashAmponlyLines", "Start", "OK"
    "User Wait" after the washing step and follow messages on the screen

  4. [  ]  template loading:
    • remove H2O from tubes in reagent positions 1,3,9,11,12 ⇒ OK
    prime air 1,3,9,11,12
    • load reagents in positions 1,3,9,11,12 ⇒ Cancel
    • remove liquid from the washing bridge [1][26 · 40µl/min · 120µl]
    • remove washing bridge
    • resume the recipe ⇒ OK
    • load Flow Cell, tube strip holder, hybridization manifold, strip #A (HT1 hybridization buffer) ⇒ OK
    CHECK!!! that all channels behave similarly
    [60µl/min · 120µl]
    • strip #B (template) ⇒ OK (time for heating),
    96°C · 1°C/s
    [15µl/min · 75µl] 5 min
    [100µl/min · 10µl]
    wait 30s
    40°C · 0.05°C/s (time for cooling)
    • strip #C (HT2 wash buffer) ⇒ OK
    [15µl/min · 75µl] 5 min
    40°C · 1°C/s
    • strip #D (APM1 ampl premix) ⇒ OK
    [15µl/min · 70µl] 5 min
    • strip #E (HFE extension mix) ⇒ OK
    [60µl/min · 95µl] 1,5 min
    74°C · 1°C/s
    wait 90s
    20°C · 1°C/s
    • strip #F (0.1N NaOH) ⇒ OK
    [60µl/min · 75µl]
    • strip #G (HT2 wash buffer) ⇒ OK
    [60µl/min · 75µl]
    60°C · 1°C/s
    • strip #0 (empty tube) ⇒ OK
    [15µl/min · 20µl]

  5. [  ]  install amplification manifold and check (IMPORTANT!) liquid flow in all channels:
    • remove hybridization manifold (put it on clean surface), connect amplification manifold ⇒ Cancel
    • manually prime line [2]3 (HT2 Wash Buffer)
    • pump HT2 (Wash Buffer) and check liquid flow in all channels [3 · 60µl/min · 100µl]
    • pump small air gap [26 · 40µl/min · 10µl]

  6. [  ]  cluster amplification:
    • resume the recipe ⇒ OK
    • "Check for proper flow through the Flow Cell and manifold" ⇒ OK;
    prime reagents 1,3,9,11,12
    35 cycles
     #9(AT1 formamide) [30µl/min · 28µl]

     #11(APM1 ampl. premix) [30µl/min · 28µl]
     #1(AMX1 ampl. mix) [30µl/min · 36µl]

    #3 (HT2 wash buffer) [30µl/min · 140µl]
    20°C · 1°C/s
    #12 (HT1 hybridization buffer) [15µl/min · 95µl]
    20°C · 1°C/s
  7. [  ]  check remaining volumes in the tubes, fill in the Cluster generation check list
  8. there are two possibilities to proceed:




[править] FlowCell storage

  1. air gap [26 · 30µl/min · 40µl];
  2. unclip "reagent delivery port" and "central clamp" of amplification manifold;
  3. remove FC and accurately clean it with water and lens cleaning tissue;
  4. put Flow Cell in 50ml tube with HT1 hybridization buffer (original storage tube), store at +4°C;



[править] CS wash

  1. wash amplification and hybridization manifolds (see "Washing of manifolds");
  2. lines washing should be perfomed with washing bridge attached;
  3. pour fresh water in water-tubes of the used positions X = 1,3,9,11,12:
    • remove tube with reagent
    • air gap [X · 240µl/min · 30µl], during the air pumping rinse the end of the tubing using washing bottle
    • attach tube with water

  4. in the recipe you used ("SR_Amplification_only_v7" or "PE_Amplification_only_v7" select the "WashAmponlyLines" step, "Start", "OK", ~10min;
  5. close the program; turn off computer, turn off CS.



[править] Reagents from the CS

  1. keep reagents 3 (HT2 wash buffer) and 12 (HT1 hybridization buffer):
    • write the date and number of the FC on the tubes
    • store at 4°C
  2. other reagents may be thrown away




[править] Notes

  1. to minimize dirt around CS it is better to leave as little as possible liquid in the manifolds you want to detach. So, remove liquid completely from the washing brifge and prime air for amplification and hybridization manifolds
  2. quite often the flow through the amplification manifold is not even in the beginning. It is better first to check the flow with some HT2 (Wash Buffer). This step is already inserted by Illumina in the recipe "PE_2P_R1Prep_Linearization_CombinedBlocking_LONG_PrimerHyb_v7". In other protocols in is necessary to perform the flow check manually either with Hybridization or wash buffer.
  • time required for separate steps:
steptime
reagents preparation30min
instrument wash15min
strip #A2min
strip #B25min
strip #C5 min
strip #D5 min
strip #E5 min
strip #F1 min
strip #G2 min
amplification2.5 hours
CS wash20 min


  • volumes of reagents after reaction (per tube):
positionreagentinitial volumeshould remainused
#AHT1 (hybridization buffer)140µl20µl120µl
#Btemplate120µl35µl85µl
#CHT2 (Wash Buffer)100µl25µl75µl
#DAPM1 (ampl.premix)100µl30µl70µl
#EHFE (extention mix)120µl25µl95µl
#F0.1 N NaOH100µl25µl75µl
#GHT2 (Wash Buffer)100µl25µl75µl
#1AMX1 (Amplification Mix)12.5ml3ml9.5ml
#3HT2 (Wash Buffer)110ml7ml4ml
#9AT1 (100% Formamide)9.5ml2.2ml7.3ml
#11APM1 (AMX1 Premix)11ml2.5ml8.5ml
#12HT1 (Hybridization Buffer)31ml24ml7ml


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