Home Second-generation sequencing > MP-long library Rambler's Top100

MP-library for long reads preparation

Equipment and consumables
DNA preparation
Primary size-selection
End repair
Ligation of the LMP CAP adapters
Secondary size-selection
DNA circularization
Digestion of non-circular DNA
Nick-translation
T7 exonuclease digestion
S1 nuclease digestion
End repair
A-tailing
Bind library to streptavidin beads
Adaptor ligation
Nick-translation

Solutions

Notes
 Comments
Illumina library / SOLiD librarySOLiD library / Illumina library



    Equipment and consumables

    • analytical agarose gel for sharing test;


    • preparative agarose gels for:
      • primary fractionation;
      • secondary fractionation;


    • QIAquick spin-columns (10-11 sets/libr.) to use after:
      • (for small quantities) shearing;
      • primary gel-fractionation (may be several columns);
      • end reparing;
      • ligation of CAP-adapters;
      • secondary gel-fractionation;
      • ligation of Internal adapters;
      • plasmid-safe DNase selection;
      • nick-translation;
      • T7 exonuclease digestion;
      • S1 nuclease digestion;
      • A-tailing;


    • check equipment:
      • vortex;
      • Hielscher UTR200 (or Covaris S2) sonicator (for small fragments);
      • HydroShear & Wash buffers (large fragments);
      • PCR machine;
      • balance;
      • tabletop centrifuge & picofuge;
      • rotator at NT;
      • magnetic stand;


    • check, that you have:
      • powder-free gloves;
      • H2O;
      • LoBind tubes & tips;




    DNA preparation

    • prepare analytical agarose gels for (i) DNA analysis & (ii) sharing test;

    1. check on 1% TAE gel ~0.1-0.5µg aliquote of DNA:
      • integrity;
      • quantity (use plasmid DNA as a control);
      • RNA contamination;
    2. if necessary, perform RNase digestion;
    3. share DNA on Hielscher UTR200 (or Covaris S2) for small (0.5-1kb) or on Hydroshare for large (>1kb) fragments;
    4. check shearing by electrophoresis in normal TAE gel (see table in Primary size-selection);
    5. if necessary, concentrate DNA:
    6. dilute DNA in 30µl of EB·;



    Primary size-selection

    • prepare standard agarose gel with combined wells (up to 6µg for well and 2µg for gap)
      DNA size [kb]gel concentration
      0.5-11%
      1-30.8%
      3-60.7%
    • for each library prepare labeled (#lib, #lib_a, #lib_b, ...) tubes for size fractions;


    1. run the gel;
    2. cut out the desired range as one block (#lib) and other sizes in ~100bp intervals (#lib_a, #lib_b, ...);
    3. freeze reserve bands in liquid nitrogen & store at -20°C;
    4. desired range block: QG-gel extraction, elute in 50µl (2x26µl):
       
      Gel blocknormal gel:
      3x QG & 1x Iso
      QG buffer
      Isopropanol
    5. determine DNA concentration on Qubit



    End repair (home-made set)

    • prepare 20°C water bath in foam plastic box;


    1. combine in a tube with DNA
       
      sheared DNA50µl
      10x T4 DNA ligase buff. with 10mM ATP (NEB)1x
      dNTP's mix, 25mM0.25mM
      T4 DNA pol., 3u/µl0.1u/µl
      Klenow DNA pol., 5u/µl0.03u/µl
      T4 PNK, 10u/µl0.3u/µl
      H2OmQ
    2. 30min 20°C in water bath;
    3. QIAquick purification:
      • PB-buffer:
      • total:
      • elute with 30µl EB (2x16µl);
    4. (optional) take 0.5µg to perform methylation analysis;



    Ligation of the LMP CAP adapters

    • prepare 20°C water bath in foam plastic box;

    1. combine in the tube with DNA (100x molar excess of adapters):
       
      DNA30µl
      LMP CAP Adapter (ds), 50 pmol/µl100x excess
      2X NEB Quick Ligase buffer1x
      40x NEB Quick Ligase1x
    2. 10 min at 20°C in water bath;
    3. QIAquick purification:
      • PB-buffer:
      • total:
      • elute with 34µl EB (2x18µl);



    Secondary size-selection

    • prepare standard agarose gel with combined wells (up to 6µg for well and 2µg for gap)
      DNA size [kb]gel concentration
      0.5-11%
      1-30.8%
      3-60.7%
    • for each library prepare labeled (#lib_1, #lib_1, ...) tubes for size fractions;


    1. run the gel;
    2. excise the thin bands (do not take DNA from the left/right borders of the band);
      3. Other reactions should be performed in parallel for individual band
    3. freeze reserve bands in liquid nitrogen & store at -20°C;
    4. first priority bands: QG-gel extraction, elute in 30µl (2x16µl):
       
      Gel blocknormal gel:
      3x QG & 1x Iso
      QG buffer
      Isopropanol
    5. determine DNA concentration on Qubit




    DNA circularization

    • prepare 20°C water bath in foam plastic box;


       0.6kb0.8kb1-2kb2-3kb3-4kb4-5 kb5-6 kb
      Opt. conc. for circularization [ng/µl]4.33.752.742.11.81.61.4
      Real concentration [ng/µl]
      DNA30µl
      Internal Adapter (ds), 2pmol/µl3x excess
      2x NEB Quick Ligase buffer1x
      40x NEB Quick Ligase1x
      H2OmQ
      PB-buffer
      volume with PB
    1. 10 min at 20°C in water bath;
    2. QIAquick purification, elute with 34µl EB (2x18µl);




    Digestion of non-circular DNA

    1. prepare plasmid-safe digestion reaction in 0.2ml tube (10u Plasmid-Safe DNase for 3µg of DNA):
       
      DNA34µl
      ATP, 25mM1.25mM
      10x Plasmid-Safe Buffer1x
      ATP dependent Plasmid-Safe DNAse, 10u/µl3.3u/µg
      H2OmQ
    2. in PCR machine: 37°C, 40 min;
    3. QIAquick purification:
      • PB-buffer:
      • total:
      • elute with 40µl EB (2x21µl);
    4. determine concentration of eluted DNA on Qubit



    Nick-translation

    1. chill the reaction on ice for 5 min before adding DNA polymerase I:
       
      circularized DNA40µl
      10x NEB Buffer 21x
      dNTP mix, 25mM0.25mM
      H2OmQ
      DNA polymerase I, 10u/µl 
    2. to obtain flanks mix thoroughly, incubate at 0°C for
    3. QIAquick purification:
      • PB:
        • // total:
      • elute with 60µl EB (2x31µl);



    T7 exonuclease digestion

    1. combine:
       
      DNA60µl
      10x NEB Buffer 41x
      T7 exonuclease, 10u/µl 
      H2OmQ
    2. mix thoroughly, incubate at 37°C for 30 min;
    3. QIAquick purification:
      • PB:
        • // total:
      • elute with 60µl EB (2x31µl);



    S1 nuclease digestion

    1. dilute S1 nuclease to 1u/µl in Dilution buffer;
    2. combine:
       
      DNA60µl
      10x S1 Nuclease Buffer1x
      NaCl, 5M150mM
      MgCl2, 100mM10mM
      S1 nuclease, 1u/µl 
      H2OmQ
    3. mix thoroughly, incubate at 37°C for 30 min;
    4. immediately proceed with QIAquick purification:
      • PB:
        • // total:
      • elute with 40µl EB (2x21µl);
    5. determine DNA concentration on Qubit;



    End repair (home-made set)

    • prepare 20°C water bath in foam plastic box;


    1. combine in a tube with DNA
       
      S1 digested DNA40µl
      10x T4 DNA ligase buff. with 10mM ATP (NEB)1x
      dNTP's mix, 25mM0.25mM
      T4 DNA pol., 3u/µl0.1u/µl
      Klenow DNA pol., 5u/µl0.03u/µl
      T4 PNK, 10u/µl0.3u/µl
      H2OmQ
    2. 30min 20°C in water bath;



    A-tailing

    1. QIAquick purification:
      • PB:
        • // total:
      • elute with 34µl EB (2x18µl);
    2. combine in 0.2ml tube:
      • DNA: 34µl
      • 10x Klenow buffer:
      • dATP, 1mM:
      • Klenow 3'->5' exo minus, 5u/µl:
      • H2O, mQ:
    3. in PCR machine:
      • 37°C, 30 min;
      • 65°C, 20 min;
      • 4°C, hold;



    Bind library to streptavidin beads



    1. stop the reaction by adding:
    2. stop the reaction by adding:

      3. prewash beads
    3. vortex Dynal MyOne C1 streptavidin beads and put 90µl into 1.5ml LoBind tube;
    4. add 500µl 1x BWB (Bead Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    5. add 500µl 1x BSA:
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    6. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;

      7. bind library
    7. add library solution in 1x SSB:
      • vortex (20 sec);
      • rotate 30 min at NT;
      • pulse-spin;
      • magnetic rack (1 min) & remove supernatant;

      8. wash library
    8. add 500µl 1x BWB (Bead Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • transfer into a new LoBind tube & vortex (20 sec);
      • pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    9. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    10. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • transfer into a new LoBind tube & vortex (20 sec);
      • pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    11. add 500µl of 1x Quick Ligase Buffer:
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    12. add 97.5µl of 1x Quick Ligase Buffer:
      • vortex (20 sec) & pulse-spin;




    Adaptor ligation

    • prepare 20°C water bath in foam plastic box;


    1. combine in the tube with PMP-bind library (10x molar excess of adapters):
      PMP suspension97.5µl
      Adapter mix, 13pmol/µl
      Quick ligase ???u/µl2.5µl
    2. mix accurately (do not vortex! do not spin!);
    3. 20 min at 20°C in water bath;


      4. wash beads
    4. add 500µl 1x BWB (Bead Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    5. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    6. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • transfer into a new LoBind tube & vortex (20 sec);
      • pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    7. two times EB-wash:
        ◊  ◊   add 500µl of EB, vortex (20 sec) & pulse-spin;
        ◊  ◊   magnetic rack (1 min) & remove supernatant;
    8. add 30µl of EB;
    9. vortex (20 sec), pulse-spin & store beads at +4°C;
    1. combine in the tube with PMP-bind library (30x molar excess of adapters):
       
      PMP suspension97.5µl
      P1 adapter, 50 pmol/µl
      P2 adapter, 50 pmol/µl
      Quick ligase ???u/µl2.5µl
    2. mix accurately (do not vortex! do not spin!);
    3. 20 min at 20°C in water bath;


      4. wash beads
    4. pulse-spin;
    5. magnetic rack (1 min) & remove supernatant;



      6. wash beads
    6. add 500µl 1x BWB (Bead Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    7. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    8. add 500µl 1x Bi-buf (Bind & Wash Buffer):
      • vortex (20 sec) & pulse-spin;
      • transfer into a new LoBind tube & vortex (20 sec);
      • pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    9. add 500µl 1x NEBuffer 2:
      • vortex (20 sec) & pulse-spin;
      • magnetic rack (1 min) & remove supernatant;
    10. add 96µl of 1x NEBuffer 2:
      • vortex (20 sec) & pulse-spin;


    Nick-translation

    • prepare 16°C water bath in foam plastic box;
    • cool magnetic rack in ice;


    1. combine in the tube with PMP-bind library:
      componentvolume
      beads with library96µl
      dNTP, 25mM2µl
      DNA Polymerase I, 10u/µl2µl
    2. mix accurately (do not vortex! do not spin!);
    3. 16°C, 30min (mixing every 5min);
    4. pulse-spin;
    5. cooled magnetic rack (1 min) & remove supernatant;
    6. two times EB-wash:
        ◊  ◊   add 500µl of EB, vortex (20 sec) & pulse-spin;
        ◊  ◊   magnetic rack (1 min) & remove supernatant;
    7. add 30µl of EB;
    8. vortex (20 sec), pulse-spin & store beads at +4°C;


    This is unamplified library. Before Cluster preparationemulsion PCR it is necessary to perform Library amplificationLibrary amplification.




      Solutions

      Streptavidin Binding Buffer, 2x

      conc.stock1ml25ml
      Tris-HCl, pH7.510mM1M10µl250µl
      EDTA1mM0.5M2µl50µl
      NaCl2M5M400µl10ml
      H2O mQ488µl12.2ml
      • make 1ml aliquotes;
      • store at +4°C;


      UDG Reaction Buffer, 10x

      conc.stock1ml5ml
      Tris-HCl, pH8200mM1M200µl1ml
      EDTA10mM0.5M20µl100µl
      DTT10mM0.5M20µl100µl
      H2O mQ760µl3.8ml
      • make 250µl aliquotes;
      • snap freeze in liquid nitrogen;
      • store at -20°C;


      1x Quick Ligase Buffer

      conc.stock1 lib5lib
      Quick Ligase Buffer1x2x300µl1.5ml
      H2O mQ300µl1.5ml
      • prepare just before use


      1x NEBuffer 2

      conc.stock1 lib5lib
      NEBuffer 21x10x60µl0.3ml
      H2O mQ540µl2.7ml
      • prepare just before use


    Notes

    • use low-binding tubes (Eppendorf) in the protocol;


    • library:
      • stock library solution should be stored at -20°C at concentration >5ng/µl;
      • the size of fragments of the M-P library should be 156bp;


    • DNA fractionation on agarose gel:
      • do not use 2% Bio-Rad agarose for fractionation of shared DNA, it has significantly lower capacity, than normal agarose;
      • best way of running the gel: 100V for the first 5 min, 30V for the next 25 min, then 100-120V;
      • one empty well should be between marker and sample;
      • marker should occupy the whole well. It permits to estimate the band distortion (upper side runs slower, than lower side);




    Second-generation sequencing
    URL: http://seq.zbio.net
    e-mail: soldatov@molgen.mpg.de
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Last modification: 12/03/09

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