digesting dna with restriction endonuclease

Digesting DNA with restriction endonuclease

1. Mix the DNA solution and reaction buffer (Total volume: Y ml)

DNA solution X l ( 5 l) 10x reaction buffer 1/Y l ( 1 l) Restriction Enzyme 1 l ( 1 l) Add ddH2O to Y l ( 3 l)

2. Incubate at 37 for 1 hour (depend on restriction en℃zymes)

3. Argarose gel electrophoresis

Recombinant DNA Technology

Gene cloning or Molecular cloning

1. The recombinant DNA construction

2. Transformation

3. Identification

The recombinantDNA construction

Transformation

Identification

Restriction Endonucleases

Restriction Endonucleases

Restriction Endonucleases

Restriction Endonucleases

Restriction Endonucleases Nomenclature

1. Eco RI Escherichia coli R2. Hpa I Haemopophilus parainfluenzae

Palindromic sequence Types of cut ends

1. 5’ phosphate extension2. 3’ hydroxyl extension3. Blunt end

Fig 4.2 Palindromic sequence

Fig 4.3

Table 4.1

Isochizomer

Different restriction enzymes recognize different sequences

Cleave within the same target sequences

Sau 3A, Mbo I & Bam HI (5’…GATC…3’)

SalI & XhoI (5’-GTCGAC- & 5’-CTCGAG-)

Fig 4.4

Fig 4.5

Fig 4.6

Ligases E coli T4

Fig 4.7

Table 4.3

Klenow fragment

3’ to pyrimidine

3’ to guanine

XceI RCATG/Y NspI RCATG/Y BstNSI, NspI

XcmICCANNNNN/

NNNNTGG XcmI CCANNNNN/NNNNTGG  

XhoI C/TCGAG PaeR7I C/TCGAGPaeR7I, Sfr274I, SlaI, S

trI, TliI

XhoII R/GATCY BstYI R/GATCYBstX2I, BstYI, MflI, Ps

uI

XmaI C/CCGGG SmaI^ CCC/GGGCfr9I, SmaI^, TspMI, X

maCI

Enzyme Sequence Enzyme Sequence Other Isoschizomers

AanI TTA/TAA PsiI TTA/TAA PsiI

AarI CACCTGC (4/8)      

AasIGACNNNN/

NNGTC DrdI GACNNNN/NNGTC DrdI, DseDI

AatI AGG/CCT StuI AGG/CCTEco147I, PceI, SseBI, S

tuI