| 5'-C T C T T C(N)1^-3' |
|
| 3'-G A G A A G(N)4^-5' |
|
| Catalog # | E0200 |
| Concentration | 10u/ul. |
| Source | Enterobacter amnigenus RFL1104 |
| Unit Definition | 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. |
| Incubation Temperature | 37°C |
| Dilution Buffer | 10mM Tris-HCl, pH 7.4 at 25°C, 100mM potassium chloride, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. |
| Storage Buffer | 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. |
| Supplied With | 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. |
| Quality Control | |
| 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 |
|
| Ligation/Recutting Assay | |
| 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. |
|
| Enzyme Properties | |
| 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. |
|
| Thermal Inactivation | |
| 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 | |
| Lambda | 34 |
| PhiX174, M13mp18/19, pBR322 | 2 |
| pUC18/19 | , pUC57, pTZ19R/U: 3 |
|
| Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| R1625 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| E0200 | 0.5-2ul |
| Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours. |
|
| Protocol for Digestion Directly after Amplification | |
| Add | |
| PCR Reaction Mixture | 10ul (~0.1-0.5ug of DNA) |
| Nuclease free water | 18ul |
| R1625 | 2ul |
| E0200 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
