Product Catalog


Afl III
A↓*C(A,G)(T,C)GT supplied with SuRE/Cut Buffer H

Catalog Number	Pack size
11209183001	100 U (5 U/µl)	Order and Price Information

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Product Description

Sequence specificity
Afl III recognizes the sequence A↓*C(A,G)(T,C)GT and generates fragments with 5’-cohesive ends.

Source
From Anabaena flos-aquae

Compatible ends
Afl III ends are compatible with ends generated by Dsa I, Nco I and Rca I.

Isoschizomers
Afl III has no known isoschizomers.

Methylation sensitivity
Afl III is inhibited by the presence of 5-methylcytosine at the site indicated (*) on the recognition sequence.

Activity in SuRE/Cut buffer System
Buffer printed in bold face type is the buffer recommended for optimal activity:

A	B	L	M	H
50-75%	75-100%	50-75%	75-100%	100%

Activity in PCR buffer
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 20%.
Note: The PCR mix contained λ target DNA, primers, 10 mM Tris-HCl (pH 8.3, 20°C), 50 mM KCl, 1.5 mM MgCl₂ , 200 μM dNTPs, 2.5 U Taq DNA polymerase. The mix was amplfied for 25 cycles.

Incubation temperature
37°C

Unit definition
One unit is the enzyme activity that completely cleaves 1 μg λDNA in 1 h at 37°C in a total volume of 25 μl SuRE/Cut buffer H.

Heat inactivation
Afl III is not heat-inactivated by incubation at 65°C; 50% of the original activity remains after 20 min incubation at 65°C.

Number of cleavage sites on different DNAs

λ	Ad2	SV40	Φ X174	M13mp7	M13mp18	pBR322	pBR328	pUC18
20	25	0	2	3	3	1	0	1

Afl III
SuRE/Cut buffer H (10x)

Quality

Absence of nonspecific endonuclease activities
1μg λDNA is incubated for 16 h in 50 μl SuRE/Cut buffer H with an excess of Afl III. The number of enzyme units that may be included in the incubation mixture without changing the enzyme-specific digestion pattern is stated under "Endo" (on the product label).

Absence of exonuclease activity
Approx. 5 μg [³H] labeled calf thymus DNA are incubated with 3 μl Afl III for 4 h at 37°C in a total volume of 100 μl 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithioerythritol, pH approx. 7.5. The amount of radioactivity released is calculated as a percentage of input radioactivity, then divided by the number of units of enzyme. That calculated value is stated under "Exo" (on the product label).

Ligation and recutting assay
Afl III fragments obtained by complete digestion of 1 μg λDNA are ligated for 16 h at 4°C with 1 U T4-DNA ligase in 10 μl buffer that contains 66 mM Tris-HCl, 5 mM MgCl₂, 5 mM dithiothreitol, 1 mM ATP, pH 7.5 (at 20°C).
The percentages of product that can be ligated and subsequently recut with Afl III (yielding the typical pattern of λ Χ Afl III fragments) are stated under "Lig" and "Rec" (on the product label).