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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » HindIII

HindIII

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Specifications

5'-A^A G C T T-3'
3'-T T C G A^A-5'
Catalog #H4000
SourceHaemophilus influenzae Rd
Concentration 10u/ul
Unit DefinitionOne unit is defined as the amount of HindIII required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer
Supplied WithR1625: 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.
Dilution Buffer10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
Storage BufferSupplied 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 InactivationHindIII is inactivated by incubation at 80°C for 20min.
Overdigestion AssayNo detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with HindIII.
Ligation/Recleavage (L/R) AssayThe ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.
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
The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.
Stability during Prolonged IncubationA 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
Lambda6
Digestion of Agarose-embedded DNAA minimum of 5 units of HindIII is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Methylation Effects
Dam, Dcm, EcoKl, CpG: never overlaps - no effect
EcoBlMay overlap - cleavage impaired.
Recommended Protocol for Digestion
Add
Nuclease free water16ul
R1625-042ul
DNA (0.5-1ug/ul)1ul
HindIII0.5-2ul
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 Mixture10ul (~0.1-0.5ug of DNA)
Nuclease free water18ul
R1625-042ul
HindIII1-2ul
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 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.


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