|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-G^T A C-3'
3'-C A T^G-5'
Unlike RsaI, Csp6I produces DNA fragments with a 2-base 5'-extension.
Corynebacterium species RFL6
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 as a liquid in 10mM Tris-HCl, pH 7.4 at 25°C, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
R1625 Restriction Enzyme Buffer A, 10X: Supplied as a liquid in33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA, pH 7.9 at 37°C.
R1625-01 Restriction Enzyme Buffer B, 10X: Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 10mM magnesium chloride, 0.1mg/ml BSA. Incubate at 37°C
Stability during Prolonged Incubation:
A minimum of 0.1u of Csp6I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Thermal Inactivation: Csp6I is inactivated by incubation at 65°C for 20min.
Compatible Ends: FspBI, NdeI, Tru1I, VspI
Number of Recognition Sites in DNA:
pBR322, pUC57, pUC18/19: 3
Dam, Dcm, EcoKI, EcoBI: Never overlaps-no effect
CpG: May overlap-no effect
Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Csp6I.
Ligation/Recutting Assay: After 50-fold overdigestion (3u/ug DNA x 17 hours) with Csp6I more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 05uM. 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 Csp6I for 4 hours.
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.
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 12 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.