Method for constructing peripheral blood lymphocyte cDNA (complementary deoxyribonucleic acid) library of Sinkiang dolang sheep

Method for constructing peripheral blood lymphocyte cDNA (complementary deoxyribonucleic acid) library of Sinkiang dolang sheep

  • CN 103,820,864 A
  • Filed: 10/29/2013
  • Published: 05/28/2014
  • Est. Priority Date: 10/29/2013
  • Status: Active Application
First Claim
Patent Images

1. a construction process for the how unrestrained sheep peripheral blood lymphocyte in Xinjiang cDNA library, is characterized in that:

  • concrete construction process and process are as follows;

    (1) extraction and the total RNA of purifying;

    gather Xinjiang how unrestrained sheep jugular vein blood 20mL and add 2mL Sodium Citrate, separate lymphocyte, utilize Trizol method to extract total RNA, purifying is placed on-70 ℃ and

    saves backup;

    (2) synthetic cDNA the first chain;

    (1.0 μ

    are g) as template take the total RNA of 3 μ

    L, add 1 μ

    L SMARTIV oligonucleotide, 1 μ

    L CDSIII/3, PCR Primer, mix, hatch 2min for instantaneous centrifugal latter 72 ℃

    , ice bath 2min, instantaneous centrifugal after, add successively 2 μ

    L5 ×

    First-stand Buffer, 1 μ

    L DTT, 1.0 μ

    L dNTP Mix, 1.0 μ

    L Power Script Reverse Transcriptase to mix, hatch 1h for instantaneous centrifugal latter 42 ℃

    , test tube is placed on ice, stops the synthetic of the first chain;

    (3) synthetic cDNA the second chain;

    preheat carrying out 95 ℃

    before the thermal cycling of PCR instrument;

    In reaction tubes, add 2 μ

    L First-Stand cDNA, 80 μ

    L ddH 20,10 μ

    L10 ×

    Advantage2PCR Buffer, 2 μ

    L50 ×

    dNTP Mix, 2 μ

    L5 '"'"' PCR primer, 2 μ

    L CDS III/3 '"'"' PCR Primer, 2 μ

    L50 ×

    Advantage2Polymerase Mix mix, the instantaneous centrifugal PCR instrument of putting into;

    PCR reaction conditions;

    95 ℃

    of 1min, 95 ℃

    of 15s, 68 ℃

    of 6min, 22 circulations, 72 ℃

    are extended 5min, and reaction product is carried out agarose gel electrophoresis detection with 5 μ

    L;

    (4) to product digest, enzyme is cut, cross post separates and connect;

    after double-stranded cDNA is synthetic through protease K digesting, SfiI enzyme is cut and is separated and remove the cDNA that is less than 500bp with CHROMA SPIN-400 post afterwards, from 16 components of collecting, respectively get 5 μ

    L, through 150V, 10min agarose gel electrophoresis, the cDNA component that contains required fragment is merged, with the sodium acetate (3M) of 1/10 volume, 1.3 μ

    L Glyecogen (20mg/mL), the ethanol precipitation of 2.5 times of volumes 95% is connected 24h with 16 ℃

    , λ

    TriplEx2Vector carrier after spending the night;

    (5) in vitro package;

    adopt Gigapack III Gold packaging Extract to pack connector;

    From the refrigerator of-80 ℃

    , take out packaging protein, be placed between finger and dissolve, the cDNA getting after 3 μ

    L connect joins packaging protein central authorities, mixes gently, avoids producing bubble, then quick centrifugal 3-5s, 22 ℃

    of water-bath 2h;

    After hatching, add 500 μ

    L SM buffer, the chloroform of 20 μ

    L, mixes gently, the instantaneous centrifugal cell debris of removing, and 4 ℃

    of storages, are the initial library of cDNA;

    (6) evaluation of cDNA library, amplification, comprises the following steps;

    The first step, cDNA initial library titre and recombination fraction are measured;

    by the cDNA library obtaining by 10 -1, 10 -2, 10 -3, 10 -4dilution, respectively gets 1 μ

    L and 200 μ

    L Host Strains XL 1-Blue (0D 600=0.5) hatch after 15min, pave plate, calculate initial library titre;

    Second step;

    the size of identification of cdna library Insert Fragment;

    144 plaques of random picking on flat board, carry out pcr amplification take 5 '"'"' LD primer and 3 '"'"' LD primer as primer, its product is identified the size of inserting cDNA fragment after electrophoresis;

    The 3rd step;

    the amplification in library;

    with 5 ×

    10 4individual phage and 600 μ

    L Host Strains XL 1-Blue (0D 600=0.5) pave plate after hatching 15min, hatch 6h for 42 ℃

    ;

    Then every plate reclaims phage and bacterial suspension after adding a SM buffer4 ℃

    shake of 9mL to wash 16h, add 5% (V/V) of chloroform to final concentration, after mixing in room temperature 15min, 2600g/min, after centrifugal 10min, 7%DMSO for supernatant (V/V) mixes rear packing and is placed in-70 ℃

    of preservations;

    The 4th step;

    cDNA amplification library titer determination presses 10 by the cDNA library after amplification -1, 10 -2, 10 -3, 10 -4dilution, respectively gets 1 μ

    L and adds 200 μ

    L Host Strains XL 1-Blue (0D 600=0.5) hatch after 15min, pave plate, calculate the cDNA library titre after amplification.

View all claims
    ×
    ×

    Thank you for your feedback

    ×
    ×