BglII (Restriction Enzyme)

REF: EG15591S

Recognition & Cleavage

Recognition site: 5′-AGATCT-3′ (palindromic)

5′ ... A^▼G A T C T ... 3′
3′ ... T C T A G_▲A ... 5′

Type: II restriction endonuclease · Common use: cloning, verification, and mapping

20 µl plasmid: 15 µl H₂O, 2 µl 10× buffer, 1 µg DNA, 1 µl BglII
30 µl PCR: 16 µl H₂O, 3 µl 10× buffer, 0.2 µg DNA, 1 µl BglII (if unpurified PCR, reduce buffer to 2 µl)
50 µl genomic: 30 µl H₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl BglII
Incubate at 37 °C: 15 min (plasmid), 15–30 min (PCR), 30–60 min (genomic)
Purify DNA after digestion (phenol/chloroform or column)

Functional test: λDNA completely digested in 15 min at 37 °C
Prolonged incubation/star activity: 3 h shows no degradation; longer incubations may cause star activity
Ligation & recutting: >95% ligation and recutting after 10× over-digest
Non-specific activity: <10% nicked/linearized plasmid after 4 h
Blue/White assay: <1% white colonies in lacZα vector test

DNA	# Sites	DNA	# Sites
λDNA	6	ΦX174	0
pBR322	0	pUC57	0
pUC18/19	0	SV40	0
M13mp18/19	1	Adeno2	11

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