Recommended Reaction Conditions
• 1X FastDigest Buffer or 1X FastDigest Green Buffer
+0.5 µM oligonucleotide.
• Incubation at 37°C.
• 1 µL of FastDigest BveI is formulated to digest up to:
– 1 µg of lambda DNA in 15 min.
– 1 µg of plasmid DNA in 15 min.
– 0.2 µg of PCR product in 60 min.
– 1 µg of genomic DNA in 20 min.
Thermal Inactivation: Incubation at 65°C for 5 min.
Methylation Effects on Digestion
Dam: never overlaps – no effect.
Dcm: never overlaps – no effect.
CpG: may overlap – cleavage impaired.
EcoKI: may overlap – effect not determined.
EcoBI: may overlap – effect not determined.
Number of Recognition Sites in DNA
λ
λλ
λ
Φ
ΦΦ
ΦX174
pBR322
pUC57 pUC18/19 pTZ19R/U M13mp18/19
Note
• At least two copies of FastDigest BveI recognition site are required
for efficient cleavage.
• Inclusion of 0.5 µM oligonucleotide with the FastDigest BveI
recognition sequence in the reaction mixture significantly improves
cleavage of plasmid DNAs, especially of those with a single
FastDigest BveI site. Still, complete cleavage of some substrates by
FastDigest
®
BveI is difficult to achieve.
• FastDigest BveI may remain associated with the cleaved DNA. This
may cause DNA band shifting during electrophoresis. To avoid
atypical DNA band patterns, use the 6X DNA Loading
Dye&SDS
Solution (#R1151) for sample preparation or heat the digested DNA
in the presence of SDS prior to electrophoresis.
CERTIFICATE OF ANALYSIS
Functional Activity Test
1 µg of lambda DNA was completely digested with 1 µL
of the enzyme in 15 minutes at 37°C in 20 µL of reaction
mixture.
Ligation and 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 oligonucleotides occured during incubation with
1 µL of FastDigest BveI for 1 hour.
Prolonged Incubation / Star Activity Assay
No detectable degradation of 1 µg of lambda DNA due
to nuclease contamination or star activity occurred during
incubation with 1 µL of FastDigest BveI for 16 hours.
Blue/White (B/W) 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.
Quality authorized by: Jurgita Zilinskiene
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