EcoRV - FAQs

I need to digest my plasmid with 2 or 3 different restriction enzymes. Can I perform a simultaneous double or even triple digestion?

If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.

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

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.

Why do I have low/no transfection efficiency with Lipofectamine PLUS reagent?

1. Order of reagent addition may have been incorrect. Be sure to mix DNA with PLUS Reagent in dilution medium first, then combine this mixture with diluted Lipofectamine Reagent.

2. Diluted DNA or PLUS Reagent was not mixed well before pre-complexing. Be sure to mix DNA well when diluting before adding PLUS Reagent or vice versa. The order of addition of DNA and PLUS Reagent to the dilution medium for pre-complexing has no effect on the transfection activity.

3. No Lipofectamine Reagent was used. Ensure that DNA-PLUS Reagent complex is mixed and incubated with diluted Lipofectamine Reagent before adding to cells.

How should restriction enzymes be stored?

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.