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View additional product information for EcoRI - FAQs (15202013)
7 product FAQs found
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.
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.
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.
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.
Star activity can be seen with many enzymes. See below for a list of some enzymes for which star activity has been reported. Some suggested causes for star activity include high glycerol concentrations, incorrect NaCl concentrations, and Mn2+ being substituted for Mg2+.
Some known enzymes that can exhibit star activity: AdeI, AfaI, AloI, Alw26I, ApoI, AseI, AvaI *, AvaII, BamHI *, BanI, BanII, BceAI, BclI, BfmI, BfuI, BglI, Bme216I, BplI, Bpu10I, BseXI, Bsh1285I, Bsh1365I, BshTI, Bsp68I, Bsp143I, Bsp143II, BspMI, BsrBRI, BssHII, BstI, Bst1107I, BstEII, BstPI, BstXI, BsuRI, CeqI, CfrI, Cfr9I, Cfr10I, CviJI 2843, Eam1105I, Eco31I, Eco32I, Eco57I, Eco72I, Eco105I, EcoKMcrBC, EcoO65I, EcoRI *, M.EcoRI, EcoRV *, EcoT22I, EheI, FbaI, HaeIII *, HhaI *, HindIII *, HinfI *, HpaI *, HphI, KspAI, MamI, MbiI, MboII, MluI *, Mph1103I, MspA1I, MunI, MvaI, NciI, NcoI *, NcuI, NdeI *, NgoMIV, NheI *, PagI, PauI, PdmI, PmeI, PpiI, PshBI, PstI *, PsuI, PvuII *, RsrI, SacI, SalI*, Sau3AI, ScaI *, SchI, SdaI, SduI, SfiI, SgfI, SgrAI, SmaI *, SpeI *, Sse838, SspI *, SstI *, SwaI, TaqI *, TatI, Tth111I, TthHB8I, Van91I, XapI, XbaI * * sold by Thermo Fisher Scientific
This "extra" band may be due to star activity of the EcoRI enzyme. The recognition site for EcoRI is GAATTC. In pCDNA3.1(+), the EcoRI site is at position 952-957. However, there is a site at 1722-1727 with the sequence GAATTA. This is presumed to be the star site, and if EcoRI exhibits star activity, a 770 bp fragment will be observed.
For pCDNA3.1(-) the EcoRI site is at 954-959, and the GAATTA is at 1721-1726. This produces a 767 bp fragment if star activity occurs. Similar bands may occur with any other vector that contains the BGH polyadenylation region, since the GAATTA occurs within this defined region.
Please use our DoubleDigest Calculator linked below: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html