Equipamentos para Laboratórios | Equipamento para Laboratório

Recognition Site:

Source:
A E. coli strain that carries the AflIII gene from Anabaena flos-aquae (CCAP 1403/13f).

Reagents Supplied:
NEBuffer 3
BSA

Enzyme Properties
Activity in NEBuffers:
NEBuffer 1:        25%
NEBuffer 2:        75%
NEBuffer 3:        100%
NEBuffer 4:        50%

When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.

Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Not sensitive
More information about: Methylation Sensitivity

Heat Inactivation:
80°C for 20 minutes

Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes

Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 3
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at 37°C.

1X NEBuffer 3:
50 mM Tris-HCl
100 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Concentration:
5,000 units/ml

Unit Assay Substrate:
λ DNA

Storage Conditions:
20 mM Tris-HCl
500 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C

Storage Temperature:
-20°C

Diluent Compatibility:
Diluent B


Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

Ligation and Re-cutting:
After a 5-fold overdigestion with AflIII, > 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut with AflIII.


 

16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA and 50 units of AflIII incubated for 16 hours at 37ºC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.

 

Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of AflIII with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.2% of the total radioactivity.


 

Reagents Sold Separately


NEBuffer 3
BSA

AflIII FAQ

Q1: Do degenerate recognition sites need to be palindromic?
A1: Most restriction enzyme recognition sites are palindromic and include only specified base pairs (i.e., EcoRI recognizes GAATTC). However, some enzymes have degenerate sites, meaning that they contain one or more base pairs that are not specifically defined (i.e., BsrFI recognizes RCCGGY, where R= A or G and Y= C or T). For degenerate enzymes, any base represented by the single letter code may be present at either location in the recognition site for cleavage to occur. For example, BsrFI recognizes all of the following sequences: ACCGGC, ACCGGT, GCCGGC, GCCGGT.