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
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