plants dna isolation-barcoding

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laboratory protocol

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  • 110125

    : DNA BARCODING

  • 1)

  • H DNA barcoding 3 .

    . ( )

    B. : DNA , ( S) PCR ,DNA sequencing editing ( , final barcode)

    . B : / , (DNA) (barcode library), , .

    A B

  • H , , ? DNA barcode, ,T/U,G,C (bar) (barcode) , - DNA ( S) , ( ,base pairs) . DNA barcode (a -sequence) . 2 / barcode .

    DNA barcodes 2 .

  • 2)

  • ( )

    1) A DNA , CTAB.

    2) E S, , PCR.

    3) PCR (gel electrophoresis), DNA (gel extraction).

    4) S1 ( DNA)

    5) , DNA a DNA sequencing (a DNA).

  • 1) A DNA , CTAB.

    eppendorf CTAB Seveg CTAB , eppendorf , , , DNA pellet pellet ( , CTAB) ddH20 pellet / ris HCl (OD)

  • Seveg ( 2min, 13000 rpm)

  • DNA pellet / ris (pH=8 , DNA ) 1 h, DNA (OD) . nanodrop , , ( DNA ng/l , DNA A260/A280, 2) /. (, / ris) .

  • E , (genomic),

    .

    ,, (marker)

  • 2) E S - -, PCR.

    1) DMSO ( (C)

    )2) dntps ()3) 2GC Buffer (5X) :

    DNA G,C .4) K polymerase (DNA )5) Primers (F,R) : , DNA

    , S (1,2) , primer sp6.6) ddH20

    / () eppendorf , DNA 1 . PCR , (, PCR , ) .

  • PCR

  • 3) PCR (gel electrophoresis).

    ( gel / TAE )

  • 1,2 % gel .To 1/10 DNA loading buffer ( bromophenol blue 4 kb), DNA PCR, (molecular weight size marker), (control) ( DNA), ( gel UV , ). gel , simple loading buffer (/ ). K , , .

  • GEL (PCR 5 )

  • GEL (PCR 3 )

  • 3) DNA gel (gel extraction) kit

    gel UV , DNA gel ( S 1,2). K gel DNA eppendorf. DNA kit NucleoSpin Gel and PCR Clean-up.

    K GEL T UV

  • : 1,3, DNA (spin column)

    Kit gel - extraction

  • DNA (spin column) (silica membrane)

  • 4) S1 DNA.

    A) LIGATION ( , insert )

    ECORI 2 ,E SERT (ITS) pGem T Easy( corI , , DNA -insert , lacZ. insert 4 ).

  • NSERT

    .coli (competent) CaCl2 (Ca) (Cl) .O insert (ITS) 10 l max, competent E.coli o (E.coli) . competent E.Coli (-80 C), LB, , 80 mM CaCl2, 100 l.

  • ) E.coli

  • ( ) (LB ).

    mini - prep

    (OVERNIGHT)

  • 5) , DNA a DNA sequencing.

    . iling prep

    mini-prep . ( )

    2 -

    2 LB 24h

    (overnight) .

    2 .

    A competent E.coli

    DNA, coRI .

    boiling prep,

    ,

    Kit Nucleospin plasmid DNA purification

    (plasmid DNA + ITS), DNA

    Sequencing.

    , coRI

    .

  • : A1, A2, A3, A4, AE DNA (spin column)

    . Quia Prep DNA kit( a DNA sequencing).

    Kit plasmid DNA purification

  • A DNA SEQUENCING