Reagents

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problems/questions

 Problem list:

  • table should be inserted type of run (51bp, 101bp, etc) / number of kits required
  • table: type of run / when to refill the reagent containers (at which cycle) / which containers for which position


Содержание


Procedure is the same for single read, PE reads 1 & 2

[править] Preliminary

  • required kits: TrueSeq SBS v5.0 (-20°C and 4°C boxes);
  • decide, how many sequencing kits would you need

insert table: type of run/number of kits

  • IMR (DNA polymerase): take out from the kit, spin down, mix by finger tapping, spin down again, leave at -20°C
  • thaw reagents in a beaker filled with room temperature water. When only faint ice pieces are noticeable, place solutions on ice:
IMR    Incorporation mix
LFN    Fluorescent nucleotides mix
SMX    Scanning mix

separately:

CLM    Cleavage mix
NB! always keep CLM away from other components, change gloves every time you deal with CLM
  • take out from 4°C:
PR1    High salt buffer
PR2    Incorporation buffer
PR3    Cleavage buffer
PW1    Wash buffer
  • record the lot numbers of reagents in the check list.


[править] Prepare

NB! when prepared, store all components on ice
  1. prepare incorporation mix:
    • add to IMR tube whole content of:


    nick reagent volume
    LFNFluorescent nucleotides mix3,52ml
    HDPDNA polymerase330µL


    • mix by inverting five times;
    • filter IMR;
  2. mix PR1, PR2, PR3, SMX by inverting;
  3. filtrate SMX;
  4. sign tubes/bottles with a reagent position number and record their weights in the check list;

    position nick reagent
    1IMR Incorporation mix
    2PW1 Wash buffer
    3 SMX Scanning mix
    4 PR1 High salt buffer
    5PR2 Incorporation buffer
    6 CLM Cleavage mix
    7 PR3 Cleavage buffer


  5. separately:
    • mix CLM by inverting;
    • centrifuge CMX36: 1min, 1000xg, NT;
    • filter CMX36;
    • sign tube, record weight;
    • wash/change gloves.

  6. if necessary fill up the 150ml bottles: insert table: type of run/reagents
Источник — «http://molbiol.ru/wiki/Reagents»


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