Technical Data
Csp6I (RsaI)
Molecular Biology Storage: -20CShipping: 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


Unit Definition:
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37C in 50ul of assay buffer.

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 7.4 at 25C, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.

Supplied With:
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 37C.

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 37C
Enzyme Properties:
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 37C.
Thermal Inactivation: Csp6I is inactivated by incubation at 65C for 20min.
Compatible Ends: FspBI, NdeI, Tru1I, VspI
Number of Recognition Sites in DNA:
Lambda: 113
PhiX174: 11
pBR322, pUC57, pUC18/19: 3
pTZ19R/U: 2
M13mp18/19: 19
Methylation Effects:
Dam, Dcm, EcoKI, EcoBI: Never overlaps-no effect
CpG: May overlap-no effect
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 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
R1625-01: 2ul
DNA (0.5-1ug/ml): 1ul
C7946: 0.5-2ul
Mix gently and spin down for a few seconds. Incubate at 37C for 1-16 hours.

Protocol for Digestion Directly after Amplification:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
R1625-01: 2ul
C7946: 1-2ul
Mix gently and 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 at least 12 months at -20C. 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.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.