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FastDigest® Restriction Enzymes

FastDigest® Mva1269I

Product information

Reaction conditions

Reaction temperature	Digestion time with 1µl of FastDigest® enzyme, min				bp from end of DNA required for complete digestion	Thermal inactivation	Incubation time without star activity, hours
	Lambda, 1µg/20µl	Plasmid DNA, 1µg/20µl	PCR product, ~0.2µg/30µl	Genomic DNA, 1µg/10µl
37°C	5	5	5	5	3	65°C, 5 min	16

Lambda DNA
1.0% agarose
46 cleavage sites

Formulation

1 μl of enzyme (1 FDU) cleaves 1 μg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.

Recommended Reaction Conditions

1X FastDigest® buffer or 1X FastDigest® Green buffer at 37°C.
1 µl of FastDigest® Mva1269I is formulated to digest up to:
– 1 µg of lambda DNA in 5 min
– 1 µg of plasmid DNA in 5 min
– 0.2 µg of PCR product in 5 min
– 1 µg of genomic DNA in 5 min, or 5 µg of genomic DNA in 30 min.

Isoschizomers

Search for commercial isoschizomers using REsearch™.

Methylation Effects

Dam: never overlaps – no effect.
Dcm: never overlaps – no effect.
CpG: may overlap – no effect.
EcoKI: never overlaps – no effect.
EcoBI: may overlap – effect not determined.

Methylation type	Sequence	Cleavage effect
CpG	5'...GAATGm5C G...3' 3'...CTTAC Gm5C...5'	No effect
CpG	5'...m5C GAATGC...3' 3'... Gm5CTTACG...5'	No effect

Number of recognition sites in DNA molecules

Lambda	ΦX174	M13mp18/19	pBR322	puc18/19	pUC57
46	4	1	1	0	1
pTZ19R/U	pTZ57R	pBluescriptIIKS(-/+)	pBluescriptIISK(-/+)	pACYC177	pACYC184
0	1	0	0	2	3

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Fast Digestion of DNA

1. Prepare the reaction mixture at room temperature in the order indicated:

   Component	Volume
   	Plasmid DNA	Unpurified PCR product	Genomic DNA
   Water, nuclease-free*	15 µl	17 µl	30 µl
   10X FastDigest® buffer or 10X FastDigest® Green buffer	2 µl	2 µl**	5 µl
   DNA*	2 µl (up to 1 µg)	10 µl (~0.2 µg)	10 µl (5 µg)
   FastDigest® enzyme	1 µl	1 µl	5 µl
   Total volume	20 µl	30 µl	50 µl

2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 5 min.***
4. Inactivate the enzyme (optional).***

Note

* The volume of water should be corrected to keep the indicated total reaction volume. The volume of DNA can be scaled up to 10 µl or down to 0.5 µl depending on the DNA concentration.
** Only 2 µl of 10X FastDigest® buffer is required for unpurified PCR product in a 30 µl reaction volume.
*** See the Certificate of Analysis for enzyme and substrate specific incubation time and enzyme inactivation conditions.

Reaction Set-up for Digestion of Multiple DNA Samples

1. Pipette 2 µl of each DNA sample* into labeled tubes.
2. Prepare a master mix for n+1 samples.
   Example of master mix (for 10 samples of plasmid DNA):

   Water, nuclease-free	(10 + 1) x 15 µl = 165 µl
   10X FastDigest® buffer or 10X FastDigest® Green buffer	(10 + 1) x 2 µl = 22 µl
   FastDigest® enzyme	(10 + 1) x 1 µl = 11 µl

3. Add 18 µl of master mix into tubes containing DNA

Note

* The volume of DNA can be scaled up to 10 µl or down to 0.5 µl depending on the DNA concentration. The volume of water and master mix should be corrected to keep the indicated total reaction volume.

Double and Multiple Digestion of DNA

FastDigest® enzymes allow simultaneous digestion of DNA with two or more enzymes in one digestion reaction.

• Use 1 µl of each enzyme and scale up the reaction conditions appropriately.
• The combined volume of all added enzymes should not exceed 1/10 of the total reaction volume.
• If enzymes require different incubation temperature, perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.

Scaling up DNA Digestion Reaction

DNA	1 µg	2 µg	3 µg	4 µg	5 µg
FastDigest® enzyme	1 µl	2 µl	3 µl	4 µl	5 µl
10X FastDigest® buffer or 10X FastDigest® Green buffer	2 µl	2 µl	3 µl	4 µl	5 µl
Total volume	20 µl	20 µl	30 µl	40 µl	50 µl