遗传与分子生物学实验 (16).pdf

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1、PRODUCT INFORMATION EcoRI#ER0271 5000 U Lot:_ Expiry Date:_ 5.G A A T T C.3 3.C T T A A G.5 Concentration:10 U/L Source:E.coli that carries the cloned ecoRIR gene from Escherichia coli RY13 Supplied with:2x1 mL of 10X Buffer EcoRI 1 mL of 10 x Buffer Tango Store at-20C In total 4 vials.BSA included

2、RECOMMENDATIONS 1X Buffer EcoRI(for 100%EcoRI digestion)50 mM Tris-HCl(pH 7.5),10 mM MgCl2,100 mM NaCl,0.02%Triton X-100,0.1 mg/mL BSA.Incubation temperature 37C.Unit Definition One unit is defined as the amount of EcoRI required to digest 1 g of lambda DNA in 1 hour at 37C in 50 L of recommended re

3、action buffer.Dilution Dilute with the Dilution Buffer(#B19):10 mM Tris-HCl(pH 7.4 at 25C),100 mM KCl,1 mM EDTA,1 mM DTT,0.2 mg/mL BSA and 50%glycerol.Double Digests Thermo Scientific Tango Buffer is provided to simplify buffer selection for double digests.98%of Thermo Scientific restriction enzymes

4、 are active in a 1X or 2X concentration of Tango Buffer.Please refer to to choose the best buffer for your experiments.1X Tango Buffer:33 mM Tris-acetate(pH 7.9 at 37C),10 mM magnesium acetate,66 mM potassium acetate,0.1 mg/mL BSA.Rev.9.Storage Buffer EcoRI is supplied in:10 mM potassium phosphate (

5、pH 7.4 at 25C),300 mM NaCl,1 mM EDTA,1 mM DTT,0.2 mg/mL BSA,0.15%Triton X-100 and 50%glycerol.Recommended Protocol for Digestion Add:nuclease-free water 16 L 10X Buffer EcoRI 2 L DNA(0.5-1 g/L)1 L EcoRI 0.5-2 L*,*Mix gently and spin down for a few seconds.Incubate at 37C for 1-16 hours*.The digestio

6、n reaction may be scaled either up or down.Recommended Protocol for Digestion of PCR Products Directly after Amplification Add:PCR reaction mixture 10 L (0.1-0.5 g of DNA)nuclease-free water 18 L 10X Buffer EcoRI 2 L EcoRI 1-2*,*Mix gently and spin down for a few seconds.Incubate at 37C for 1-16 hou

7、rs*.*This volume of the enzyme is recommended for preparations of standard concentrations(10 U/L),whereas HC enzymes(50 U/L)should be diluted with the Dilution Buffer to obtain 10 U/L concentration.*See Overdigestion Assay.Thermal Inactivation EcoRI is inactivated by incubation at 65C for 20 min.ENZ

8、YME PROPERTIES Enzyme Activity in Thermo Scientific REase Buffers,%EcoRI B G O R Tango 2X Tango 100 0-20 NR 100 100*NR 100*Star activity appears at a greater than 5-fold overdigestion(5 U 1h).NR buffer is not recommended,because of high star activity.Methylation Effects on Digestion Dam:never overla

9、ps no effect.Dcm:never overlaps no effect.CpG:may overlap cleavage impaired.EcoKI:never overlaps no effect.EcoBI:may overlap no effect.Stability during Prolonged Incubation A minimum of 0.2 units of the enzyme is required for complete digestion of 1 g of lambda DNA in 16 hours at 37C.Digestion of Ag

10、arose-embedded DNA A minimum of 5 units of the enzyme is required for complete digestion of 1 g of agarose-embedded lambda DNA in 16 hours.Compatible Ends XapI,MunI,TasI Number of Recognition Sites in DNA X174 pBR322 pUC57 pUC18/19 pTZ19R/U M13mp18/19 5 0 1 1 1 1 1 For CERTIFICATE OF ANALYSIS see ba

11、ck page CERTIFICATE OF ANALYSIS Overdigestion Assay No detectable change in the specific fragmentation pattern is observed after an 80-fold overdigestion with EcoRI (5 U/g lambda DNA 16 hours).Ligation and Recleavage(L/R)Assay The ligation and recleavage assay was replaced with LO test after validat

12、ing experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.Labeled Oligonucleotide(LO)Assay No detectable degradation of single-stranded or double-stranded labeled oligonucleotides occurred during incubation with 10

13、 units of EcoRI for 4 hours.Blue/White(B/W)Cloning Assay The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.Quality authorized by:Jurgita Zilinskiene PRODUCT USE LI

14、MITATION This product is developed,designed and sold exclusively for research purposes and in vitro use only.The product was not tested for use in diagnostics or for drug development,nor is it suitable for administration to humans or animals.Please refer to for Material Safety Data Sheet of the product.2012 Thermo Fisher Scientific Inc.All rights reserved.All trademarks are the property of Thermo Fisher Scientific Inc.and its subsidiaries.