|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-C T C T T C(N)1^-3'
3'-G A G A A G(N)4^-5'
Enterobacter amnigenus RFL1104
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.
10mM Tris-HCl, pH 7.4 at 25°C, 100mM potassium chloride, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
Supplied as a liquid in10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
R1625: Restriction Enzyme Buffer A, 10X. Provided to simply buffer selection.
Storage and Stability:
May be stored at 4°C for short-term only. For long-term storage, aliquot and 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. Further dilutions can be made in assay buffer.
Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Eam1104I
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Eam1104I, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.2uM. More than 95% of these can be recut.
Labeled Oligonucleotide Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of rEam1104I for 4 hours.
Stability during Prolonged Incubation:
A minimum of 0.5u of Eam1104I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Eam1104I is inactivated by incubation at 65°C for 20min.
Digestion of Agarose-Embedded DNA:
A minimum of 5u of Eam1104I is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Number of Recognition Sites in DNA:
PhiX174, M13mp18/19, pBR322: 2
pUC18/19:, pUC57, pTZ19R/U: 3
Protocol for Digestion:
Nuclease free water: 16ul
DNA (0.5-1ug/ml): 1ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Protocol for Digestion Directly after Amplification:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.