MP-library preparation (nick-translation)

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Illumina / SOLiDSOLiD / Illumina

Содержание


This protocol is for preparation of unamplified library. Before Cluster preparationemulsion PCR it is necessary to perform Library amplification.

[править] Circularization

[править] Sample preparation

  • prepare in advance analytical agarose gels for:
» DNA analysis
» shearing test


    [править] Sample analysis

  1. perform sample analysis;
  2. decide:
    » if sample is good for library preparation: quantity and quality
    » for samples with large amount of DNA, decide how to split experiment/reserve
    library size [kb]DNA quantity [µg]
    1-2.5kb30-60[1]
    2.5-4kb40-80[1]
    >4kb 

  3. [править] DNA purification

  4. DNA from external source[2]:
    » RNase digestion[3], check digestion on electrophoresis: 0.2µg of DNA in 0.7-1% TAE gel[4]
    » Proteinase K treatment and Phenol/Chlorophorm purification
    » NH4Ac/Ethanol precipitation
    » dilute in EB at ~0.2µg/µl
    » check DNA concentration on Qubit (BR kit)

    [править] DNA shearing

    insert auto-calculation form

  5. shear DNA
    DNA size [kb]maschine
    0.5-1Covaris S2 (better) or UTR200[5]
    >1kbHydroshear
  6. NH4Ac/"Isopropanol or Ethanol" precipitation[6];
  7. dilute DNA in EB at ~0.2µg/µl;
  8. pool and check DNA concentration on Qubit: Qubit (BR kit)


[править] Primary size-selection[7]

insert auto-calculation form prepare for each sample:

  • labeled (#lib.1, #lib.2, #lib.3 — from small sizes to large) tubes for size fractions
  • standard agarose gel (one sample — one electrophoresis) with combined wells (up to 6µg for well and 2µg for gap)[8]
  • check well leakage[9]
DNA size [kb]gel concentration [%]
0.5-11
1-30.8
3-60.7


  1. run the gel[10] untill desired fragments will reach 0.5-0.7x length of the gel[11];
  2. cut out
    » the desired range as one block
    » other smear as ~2 reserve blocks[12];
  3. freeze reserve blocks in liquid nitrogen and store at -20°C;
  4. make photo of the gel for check-list[13];
  5. large-scale QG-gel extraction:
    » estimate the amount of DNA in the desired gel block:
    » select column type and quantity:
    » elute in 30µl (2x16µl) from each column
    insert auto-calculation form
    volume500µl
    DNA1xV100µl
    QG buffer3xV300µl
    Isopropanol1xV100µl

  6. pool, determine DNA concentration on Qubit: BR kit
    » NB! to obtain ~1.5kbp library with complexity ~10? molecules, it should be >5µg DNA on this step


[править] End repair (home-made set)

  • prepare 20°C water bath in foam plastic box[14]


  1. combine in 1.7ml tube
     
    sheared DNA
    H2OmQ
    ER buffer, 9.08x1x
    ER enzyme mix, 12x~1x

  2. 30min 20°C in water bath;
  3. Proteinase K treatment and Phenol/Chlorophorm purification;
  4. add 1µl of glycogen (20µg/µl), NaCl/EtOH precipitation



[править] 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), 50pmol/µl100x excess
    NEB Quick Ligase buffer, 2x1x
    NEB Quick Ligase, 40x1x
  2. 10 min at 20°C in water bath;
  3. QIAquick purification:
    • PB-buffer:
    • // total:

    • elute with 34µl EB (2x18µl);


[править] Secondary size-selection

purpose:

  • to prevent formation of heterohybrids
  • to remove adapters, which may inhibit circularization


prepare:

  • standard agarose gel with combined wells as described in the Primary size-selection
  • for each library — labeled (#lib_1, #lib_2, ...) tubes for size fractions


  1. run the gel;
  2. excise the thin gel-blocks[15];
  3. other steps should be performed in parallel for individual gel-blocks
  4. freeze reserve bands in liquid nitrogen and store at -20°C;
  5. first priority bands: QG-gel extraction (normal gel: 3x QG, 1x Iso), elute in 30µl (2x16µl):
    Gel block
    QG buffer
    Isopropanol

  6. determine DNA concentration on Qubit;
  7. store at -20°C.



[править] DNA circularization

  • prepare 20°C water bath in foam plastic box


  1. combine in the tube with DNA:
     0.6kb0.8kb1-2kb2-3kb3-4kb4-5 kb5-6 kb
    Optimal 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
  2. 10 min at 20°C in water bath;
  3. QIAquick purification (5x PB, see table), elute with 34µl EB (2x18µl);
  4. prepare plasmid-safe digestion reaction[16]:
     
    DNA34µl
    ATP, 25mM1.25mM
    10x Plasmid-Safe Buffer1x
    ATP dependent Plasmid-Safe DNAse, 10u/µl3.3u/µg
    H2OmQ
  5. in PCR machine:
    • 37°C, 40 min
  6. 30 min 20°C in water bath;
  7. QIAquick purification:
    • PB-buffer:
    • // total:
    • elute with 40µl EB (2x21µl);
  8. determine concentration of eluted DNA on Qubit.


[править] Nick-translation

  1. chill the reaction on ice for 5 min before adding DNA polymerase I:
     
    circularized DNA40µl
    NEB Buffer 2, 10x1x
    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);


[править] T7 exonuclease digestion

  1. combine:
     
    DNA60µl
    NEB Buffer 4, 10x1x
    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);


[править] S1 nuclease digestion

  1. dilute S1 nuclease to 1u/µl in Dilution buffer;
  2. combine:
    DNA60µl
    S1 Nuclease Buffer, 10x1x
    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.


[править] End repair (home-made set)

  • prepare 20°C water bath in foam plastic box


  1. combine in a tube with DNA
    S1 digested DNA40µl
    NEB T4 DNA ligase buff. with 10mM ATP, 10x 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;


[править] A-tailing (only Illumina)

  1. QIAquick purification:
    • PB:
    • // total:

    • elute with 34µl EB (2x18µl);
  2. combine in 0.2ml tube:
    DNA34µl
    Klenow buffer, 10x
    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


[править] Library enrichment


[править] Bind library to streptavidin beads

  1. stop end-repair reaction by adding:




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

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

    wash library
  6. 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
  7. add 500µl 1x Bi-buf (Bind and Wash Buffer):
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  8. add 500µl 1x Bi-buf (Bind and Wash Buffer):
    • vortex (20 sec), pulse-spin
    • transfer into a new LoBind tube
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant


[править] Adaptor ligation

  • prepare 20°C water bath in foam plastic box


  1. add 500µl of 1x Quick Ligase Buffer:
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  2. add 97.5µl of 1x Quick Ligase Buffer:
    • vortex (20 sec), pulse-spin
  3. combine in the tube with PMP-bind library:
    PMP suspension97.5µl
    Adapter mix, 13pmol/µl10x molar excess of adapters
    P1 adapter, 50pmol/µl30x molar excess of adapters
    P2 adapter, 50pmol/µl
    NEB Quick ligase, 40x1x2.5µl

  4. mix accurately (do not vortex! do not spin!);
  5. 20 min at 20°C in water bath;

    wash beads
    • pulse-spin
    • magnetic rack (1 min), remove supernatant
  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 and Wash Buffer):
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  8. add 500µl 1x Bi-buf (Bind and Wash Buffer):
    • vortex (20 sec), pulse-spin
    • transfer into a new LoBind tube
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant


[править] Nick-translation (only SOLiD)

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


  1. add 500µl 1x NEBuffer 2:
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  2. add 96µl of 1x NEBuffer 2:
    • vortex (20 sec) and pulse-spin
  3. combine in the tube with PMP-bind library:
    beads with library96µl
    dNTP, 25mM2µl
    DNA Polymerase I, 10u/µl2µl
  4. mix accurately (do not vortex! do not spin!);
  5. 16°C, 30min (mix every 5min);
  6. pulse-spin;
  7. cooled magnetic rack (1 min), remove supernatant.


[править] Wash for storage

  1. ○○   two times EB-wash:
    • add 500µl of EB
    • vortex (20 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  2. add 30µl of EB:
    • vortex (20 sec), pulse-spin
  3. store beads at +4°C.


This is unamplified library. Before Cluster preparation it is necessary to perform Library amplification.

This is unamplified library. Before emulsion PCR it is necessary to perform Library amplification.


[править] Materials

[править] Equipment and consumables

analytical agarose gels for:

  • DNA analysis: 0.7-1% TAE
  • shearing test


preparative agarose gels for:

  • primary fractionation
  • secondary fractionation


QIAquick spin-columns (6-8 sets/libr.) to use after:

  • (for small quantities) shearing;
  • primary gel-fractionation (may be several columns);
  • end reparing;
  • methylation;
  • ligation of CAP-adapters;
  • secondary gel-fractionation;
  • ligation of Internal adapters;
  • plasmid-safe DNase selection;
  • A-tailing;


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


other:

  • powder-free gloves
  • scalpels & beaker with water to wash scalpel
  • low size-range DNA marker
  • H2O
  • LoBind tubes and tips




[править] Solutions

[править] Streptavidin Binding Buffer, 2x

conc.stock1ml25ml
Tris-HCl, pH7.510mM1M10µl250µl
EDTA1mM0.5M2µl50µl
NaCl2M5M400µl10ml
H2O mQ488µl12.2ml

  • store in 1ml aliquotes at +4°C


[править] UDG Reaction Buffer, 10x

conc.stock1ml5ml
Tris-HCl, pH8200mM1M200µl1ml
EDTA10mM0.5M20µl100µl
DTT10mM0.5M20µl100µl
H2O mQ760µl3.8ml

  • store in 250µl aliquotes at -20°C
  • snap freeze in liquid nitrogen


[править] Quick Ligase Buffer, 1x

conc.stock1 lib5lib
Quick Ligase Buffer1x2x300µl1.5ml
H2O mQ300µl1.5ml

  • prepare just before use


[править] NEBuffer 2, 1x

conc.stock1 lib5lib
NEBuffer 21x10x60µl0.3ml
H2O mQ540µl2.7ml

  • prepare just before use




[править] 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 (System??? protocol???)


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)


[править] References

  1. 1,0 1,1 if more, leave for the second try
  2. skip purification for samples with known good quality
  3. in principle, it is possible to shear DNA by ultrasound after RNase treatment w/o purification
  4. ~5cm long gel, 120V per 28cm
  5. for unclear reasons, sometimes shearing works less efficient — shearing control is obligatory
  6. Isopropanol or Ethanol precipitation: isopropanol occupies ~1/2, Ethanol — ~2/3 of the tube. Use Isopropanol when application of Ethanol leads to precipitation in several tubes.]
  7. primary size-selection helps:
    • to prevent formation of heterohybrids (if small-size border is more than "1/2 of large-size border")
    • to reduce amount of DNA, so less enzymes and adapters will be spent for library preparation
  8. there should be one empty well between sample and marker. Loading schemes for
    » one combined well per gel: _·M·_·SAMPLE·_·M·_
    » two combined wells per gel: _·M·_·SAMPLE·_·M·_·SAMPLE·_·M·_
  9. ~5µl/well of 0.5x Br.Ph. SDS-loading buffer
  10. ~10cm long gel, 120V per 28cm
  11. 1-2kbp: untill OrangeG in 1-2cm from the end of the gel
  12. reserve blocks are collected
    » (primary) for repeat of experiment in case of problems
    » as DNA source for possible future experiments
    so, reserve bands should have reasonable (similar to "desired range") size distribution and cover most of the smear. It is possible, that more than 2 reserve gel blocks would be required. Typical for 1.5kb MP-library: 1.1-1.4 / 1.4-1.7 / 1.7-2.1
  13. for gel photo: insert paper pieces into cuts
  14. important not to increase temperature, otherwise exonuclease activity of T4 polymerase will be too high
  15. do not take DNA from the left/right borders of the band
  16. 10u Plasmid-Safe DNase for 3µg of DNA


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