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Specifications

5'-A^A G C T T-3'
3'-T T C G A^A-5'
Catalog #	H4000
Source	Haemophilus influenzae Rd
Concentration	5u/ul
Unit Definition	10 units/ul. One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer
Supplied With	R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA (pH 7.9 at 37°C).
R1625-04	Restriction Enzyme Buffer E, 10X: Supplied as a liquid in 10mM Tris-HCl, pH 8.5, 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 37°C.
Storage and Dilution Buffer	Supplied as a liquid in 10mM Tris-HCl, pH 7.4 250mM KCl, 0.1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
Thermal Inactivation	HindIII is inactivated by incubation at 65°C for 20min.
Overdigestion Assay	No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with HindIII.
Ligation/Recutting Assay	After 50-fold overdigestion (3u/ug DNA x 17 hours) with HindIII, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.97M. More than 95% of these can be recut.
Labeled Oligonucleotide (LO) Assay	No detectable degradation of single- stranded or double-stranded labeled oligonucleotide was observed after incubation with 10 units of HindIII for 4 hours.
Blue/White Cloning Assay	pUC57 was incubated with 10 units of HindIII for 16 hours. After religation and transformation 0.2% of white colonies were detected.
Stability during Prolonged Incubation	A minimum of 0.1 units of HindIII is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Number of Recognition Sites in DNA	Lambda: 7: PhiX174: 0: M13mp18/19, pBR322, pUC18/19, pUC57, pTZ19R/U, pBluescriptIIKS(-/+), pACYC177, pBluescriptIISK(-/+), pACYC184: 1
Digestion of Agarose-embedded DNA	A minimum of 5 units of HindIII is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Recommended Protocol for Digestion	Add:
	Nuclease free water: 16ul
	R1625-04: 2ul
	DNA (0.5-1ug/ul): 1ul
	HindIII: 0.5-2ul
	Mix gently, spin down for a few seconds.
	Incubate at 37ºC for 1-16 hours.
Protocol for Digestion of PCR products directly after amplification	Add:
	PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
	Nuclease free water: 18ul
	R1625-04: 2ul
	MlsI: 1-2ul
	Mix gently, spin down for a few seconds.
	Incubate at 37ºC for 1-16 hours.
Storage and Stability	May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Important Note	This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.