Technical Data
H4000
HindIII
5KU
25KU
Molecular Biology Storage: -20CShipping: Blue Ice
5'-A^A G C T T-3'
3'-T T C G A^A-5'

Source:
Haemophilus influenzae Rd

Concentration: 10u/ul

Unit Definition:
One unit is defined as the amount of HindIII required to digest 1ug of lambda DNA in 1 hour at 37C 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 37C).
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 37C.

Dilution Buffer:
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.

Storage 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 80C 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/Recleavage (L/R) Assay: The 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 Incubation: A minimum of 0.1 units of HindIII is required for complete digestion of 1ug of lambda DNA in 16 hours at 37C.

Number of Recognition Sites in DNA:
Lambda: 6: M13mp18/19, pBR322, pUC18/19, pUC57, pTZ19R/U: 1: PhiX174: 0

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.

Methylation Effects:
Dam, Dcm, EcoKl, CpG: : never overlaps - no effect
EcoBl: may overlap - cleavage impaired.

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 37C 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
HindIII: 1-2ul
Mix gently, spin down for a few seconds.
Incubate at 37C for 1-16 hours.

Storage and Stability:
May be stored at 4C for short-term only. For long-term storage, store at -20C. Aliquots are stable for 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.