Product FAQ

What are the best conditions for double digests with conventional Thermo Scientific restriction endonucleases?

Answer

First, please note that we changed our restriction enzymes and buffer formulations in 2010. The REact buffers 1-10 were discontinued in favor of a smaller group of universal buffers: B, G, O, R and Tango (Y). The new buffers are not compatible with older restriction enzymes, and it is not recommended to do a double digest with an old enzyme (with REact buffer) and a new enzyme.

When performing any double digest, there may be buffer incompatibilities and enzyme steric hindrance problems. These can be avoided by performing sequential digests, separated by buffer exchange or chloroform extraction and ethanol precipitation. However, if these issues are understood and a double digest will be performed, you can evaluate enzyme combinations using our buffer compatibility chart: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/conventional-restriction-enzymes-thermo-scientific/reaction-conditions-for-restriction-enzymes.html.

Answer Id: E2929

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Product FAQ

What effects do Dam or Dcm methylase have on restriction enzyme digestion of DNA?

Answer

Certain restriction enzymes are unable to recognize and cleave at their target sites if specific adenine or cytosine residues in the sequence are methylated, and Dam and Dcm are two E. coli methylases which introduce methyl groups that affect the cutting sites of many common enzymes. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group from S-adenosylmethionine to the N6 position of the adenine residues in the sequence GATC. The Dcm methylase (encoded by the dcm gene; referred to as the Mec methylase in earlier references) methylates the internal cytosine residues in the sequences CCAGG and CCTGG at the C5 position.

To take advantage of Dam- and Dcm-sensitive restriction enzymes and get proper cleavage, plasmid DNA must be propagated in and isolated from an E. coli strain that is deficient in the endogenous Dam methylase and Dcm methylase enzymes just prior to the restriction reaction. We have one competent cell product available that is made with a dam- and dcm- strain: One Shot INV110 Chemically Competent E. coli (Cat. No. C7171-03).

Answer Id: E4005

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Product FAQ

Is it possible to perform a double digestion using an Anza restriction enzyme and a FastDigest restriction enzyme or Thermo Scientific restriction enzyme that is not available in the Anza collection?

Answer

For optimal results, we highly recommend exclusively using Anza restriction enzymes in double digestion as they all have 100% buffer compatibility. If a certain enzyme is not available in Anza format, but is available in FastDigest format or as a Thermo Scientific conventional restriction enzyme, it may be possible to perform a double digestion. For specific protocol recommendations, please contact our technical support with detailed information of the desired enzymes and DNA template.

Answer Id: E13442

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Product FAQ

How can I check the activity of a restriction enzyme?

Answer

Here are some recommendations:
1. You can verify the restriction endonuclease has activity by digesting the unit substrate (i.e., lambda or Ad-2 DNA) using the reaction conditions identified for unit definition: one unit of the restriction endonuclease should digest one µg of the unit substrate in one hour in the appropriate reaction buffer at the appropriate temperature.
2. You can test for the presence of inhibitors of restriction digestion in the sample DNA. Perform a restriction digest in which some of the sample DNA is included along with some of the unit substrate (i.e., lambda or Ad-2 DNA). If digestion of the unit substrate occurs alone but is not observed when the sample DNA is added, then a diffusible inhibitor of restriction digestion is present in the sample DNA. If digestion of the unit substrate occurs in the presence of the sample DNA, but the sample DNA is not digested, then the failure of the restriction endonuclease may be due to sensitivity of the restriction endonuclease to methylation in the sample DNA.
3. Finally, to identify any tube-specific issues like shipment or storage stability problems, you can test function by performing a side-by-side reaction with a different lot of the same Invitrogen restriction endonuclease and comparing results.

Answer Id: E4004

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Product FAQ

Why is there a number in the name of the Anza restriction enzymes?

Answer

Anza restriction enzymes contain a number preceding the name. This number provides the option to organize the enzymes in freezers numerically or alphabetically. More common enzymes tend to have a lower number.

Answer Id: E13440

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Product FAQ

Which restriction enzymes have been tested with the CloneChecker system?

Answer

Restriction endonuclease digestion of released plasmid DNA was successful with 20 of the popular enzymes used in cloning: Acc I, Ava I, BamH I, BstE II, Bgl II, Cla I, EcoR I, Hpa I, Hind III, Kpn I, Nco I, Nhe I, Not I, Sac II, Sal I, Ssp I, Sst I, Spe I, Xba I, and Xho I. In addition to single digestions with these enzymes, a number of double digests were successful as well. No enzyme tested has failed to perform properly. Note, restriction digests are only possible with plasmid released with the GREEN solution from the Restriction Enzyme Digest method.

Answer Id: E3087

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Product FAQ

What is the unit definition for Anza restriction enzymes?

Answer

- A unit is defined as the amount of enzyme required to completely digest 1 µg of substrate DNA in 50 µL of the reaction mixture in 1 hour at 37 degrees C.
- Anza restriction enzymes and buffer have been formulated such that 1 µL of Anza restriction enzyme cleaves 1 µg of substrate DNA in 15 minutes in Anza buffer.

Answer Id: E13438

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Product FAQ

Can Anza restriction enzymes digest unpurified PCR products in PCR buffer?

Answer

-Digestions in several PCR reaction buffers were tested and the Anza restriction enzymes fully digested DNA. However, not all commercially available buffers could be tested.

To obtain the best results for unpurified PCR products, we recommend the following reaction conditions:
- Up to 10 µL of PCR reaction mixture
- 1 µL of 10X Anza Buffer
- 1 µL of Anza restriction enzyme
- Up to 20 µL of H2O
- 37 degrees C, 15 min

If digested PCR product will be used for cloning, it is necessary to purify the PCR fragment prior to digestion to remove the active polymerase. Active thermophilic DNA polymerase will still be present in the PCR mixture if it is not removed by purification and may alter the ends of the cleaved DNA thereby reducing ligation efficiency.

Answer Id: E13436

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Product FAQ

Can I double digest my DNA using a Thermo Scientific conventional restriction enzyme and a FastDigest restriction enzyme?

Answer

For optimal results with fast reaction and 100% buffer compatibility, we highly recommend using FastDigest restriction enzymes in double digestion. In certain cases however, it may be possible to perform double digestion using a mix of Thermo Scientific conventional and Fastdigest restriction enzymes. For specific recommendations, please contact our technical service with detailed information about the enzymes and DNA template you plan to use.

Answer Id: E8824

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Product FAQ

What guidelines can you give me for the recommended amount of a restriction endonuclease required for complete digestion of my plasmid?

Answer

Relative enzyme activities can vary widely, and the amount used may also need to be adjusted according to the size and surrounding sequence of your target molecule. But to give a general idea of the relative activity of various enzymes, we performed a standardized digestion experiment using all of the enzymes listed below. The list indicates the number of units of enzyme that were needed for complete digest of 1 ?g supercoiled pBR322 DNA in 1 hour.

Enzyme - Units

Acc I - 4
Afl III - 0.5
Alu I - 1
AlwN I - 0.5
Ava I - 1
Ava II - 0.5 (a)
BstY I - 8
BamH I - 2
Ban II - 2
Bgl I - 8
Bsm I - 8
Cfo I - 4
Eco47 III - 8
Cla I - 1 (b)
Dde I - 1
Dra I - 4
EcoO109 I - 8
EcoR I - 0.5
EcoR II - >8 (a)
EcoRV - 2
Fsp I - 1
Hae II - 2
Hae III - 2
Hha I - 2
Hinc II - 4
Hind III - 0.5
Hinf I - 2
Hpa II - 1
Kpn2 I - 4
Mbo I - >8 (b)
Mbo II - 4 (b)
Msc I - >8 (a)
Mse I - 2
Msp I - 0.5
Nar I - 2
Nci I - 4
Nde I - 4
Nde II - >8 (b)
NgoA IV - 4
Nhe I - 2
Nsp I - 2
Nru I - 2 (b)
Psp5 II - 8
Pst I - 4
Pvu I - 2
Pvu II - 2
Rca I - 4
Rsa I - 4
Sal I - 8
Sau3A I - 2
Sau96 I - 8 (a)
Sca I - 4
Sph I - 2
Ssp I - 4
Sty I - 8
Taq I - 2 (b)
Tha I - 4
Vsp I - 0.5
Xma III - >8

(a) Sensitive to dcm methylation
(b) Sensitive to dam methylation

Notes:
- This list includes only restriction endonucleases with cleavage sites in pBR322, and not all enzymes listed are currently available for purchase from Thermo Fisher Scientific.
- In the experiments that generated this data, plasmid pBR322 was grown in E. coli RR1 and thus contained 5-methylcytosine and N6-methyladenine. Reactions contained 0.5, 1.0, 2.0, 4.0, or 8.0 units of enzyme, 1 µg of pBR322 DNA and the appropriate REACT Buffer in 20 µl. After a 1-h reaction, products were analyzed by electrophoresis on a 1% agarose gel.

Answer Id: E4148

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Product FAQ

How should restriction enzymes be stored?

Answer

Restriction endonucleases should generally be stored at -20°C in a non frost-free freezer. Enzymes should be kept on ice during use and returned to the -20°C freezer as quickly as possible.
However, please be sure to double-check the recommended storage conditions in your product manual or packaging for any special instructions.

Answer Id: E4007

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Product FAQ

Do Anza restriction enzymes contain BSA?

Answer

All Anza restriction enzymes contain BSA.

Answer Id: E13445

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Product FAQ

How can I perform large scale digestion with the FastDigest restriction enzymes?

Answer

Our general recommendation is to use 5-10 µL of FastDigest enzymes with 100 µg DNA in 500 µL and 10-20 µL of the FastDigest enzymes with 200 µg DNA in 1 mL. Incubate the reaction for 16 h (or the longest recommended incubation time if the particular enzyme is prone to star activity) at the recommended temperature. Optimal conditions may vary as complete digestion of DNA depends on the nature of the particular FastDigest restriction enzymes and the DNA substrates.

Answer Id: E8833

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Product FAQ

What is the unit or activity of FastDigest Restriction Enzymes?

Answer

Activity of FastDigest enzymes is defined as 1 µL of FastDigest enzyme cleaves1 µg of substrate DNA in 5 to 15 minutes in 20 µL of FastDigest buffer. This is in contrast to activity of conventional restriction enzymes which is defined as 1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in 50 µL of optimal buffer for an enzyme.

Answer Id: E8828

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Product FAQ

What are the suggested conditions for restriction enzyme digestions?

Answer

Conditions can vary, so it is always best to follow any protocols provided with your specific enzyme. But the majority of enzymes to have similar protocols and here are some general guidelines:

- Most restriction enzyme digestions require 2-10 units enzyme/µg DNA.
- Restriction enzyme and the appropriate reaction buffer should be added to your DNA solution, and the volume of enzyme added should generally not be more than 10% of the DNA solution volume.
- Reaction buffers (like REact) are usually provided at 10X concentration, so they should be added such that their volume will represent exactly 1/10 of the final volume of DNA + restriction components.
- Recommended incubations are usually 1-2 hours at 37 degrees C.

Answer Id: E4006

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