Recommended
buffer |
Reaction conditions |
|
Temperature |
Time |
| FastDigest® buffer
+ 0.01 mM SAM |
37°C |
5 min |
Thermal Inactivation
FastDigest® AjuI is inactivated by incubation at 65°C for
5 min.
Digestion of Plasmid DNA
1 µl of FastDigest® AjuI digests up to 1 µg of plasmid DNA in 5 min, see
Protocol for Fast
Digestion and Analysis of Plasmid DNA with FastDigest® Enzymes
and
Protocol for Fast Vector
Preparation with FastDigest® Enzymes.
Digestion of PCR Products
1 µl of FastDigest® AjuI digests ~0.2 µg of PCR product in
10 min,
see
Protocol for Fast Digestion of PCR
Products with FastDigest® Enzymes.
Digestion of Genomic DNA
1 µl of FastDigest® AjuI digests 1 µg of genomic DNA in 5 min, or 5 µg of
genomic DNA in 30 min, see
Protocol for Fast Digestion of Genomic DNA with FastDigest® Enzymes.
Ligation
and Recleavage
After overdigestion with 1 µl of FastDigest® AjuI
for 1 hour, more than 90% of DNA fragments can be ligated and recut.
| Methylation
Effects |
Dam:
Dcm:
CpG:
EcoKI:
EcoBI: |
never overlaps
never overlaps
may overlap
never overlaps
never overlaps |
- no effect.
- no effect.
- no effect.
- no effect.
- no effect. |
Note
- FastDigest® AjuI requires SAM for activity.
- Complete cleavage of some substrates with FastDigest® AjuI is difficult to
achieve.
- FastDigest® AjuI produces double-strand cuts on both sides of the
interrupted recognition site. The exact cleavage position depends on the
sequences flanking the recognition site and may shift by one base pair.
For an individual sequence, there will be a mix of the 2 cleavage
positions, with the majority of the digestion occurring at one of the
positions.
Recommendations for Double and Triple Digestions with FastDigest® Enzymes
Perform 5 minute double or triple digestions with FastDigest®
AjuI and any other FastDigest® enzyme(s) in FastDigest® buffer,
see Protocol for
Double Digestion with FastDigest® Enzymes and
Protocol for Triple Digestion with
FastDigest® Enzymes.
