User Manual - TopoGEN, Inc.
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1. but keep your electrophoresis times to less than 1 2 hrs Long run times cause band diffusion and degrade the quality of your gel results TopoGEN Inc www topogen com Protocol TG2000GKIT 6 Version E coli DNA Gyrase User Manual What reaction volumes do you recommend for these assays Reaction volumes should be 20 30 ul final volume limited by the volume that can be loaded into the wells of the agarose gel The reactions should be assembled on ice in microfuge tubes water buffer and DNA test compound and enzyme which should be added last After adding enzyme the tubes should be transferred to a heating block to initiate the reaction Does it matter whether run a large format gel or a minigel Either will work Minigels are very convenient and give reasonably fast run times The actual shape and size of the well slot is a factor in how well your bands resolve It is best to use a comb that gives a long and thin rectangular Are the termination conditions critical for detecting cleavages Yes Reactions should be incubated 30 min 37 C terminated by rapid addition of 1 10 volume of 10 SDS followed by digestion with 50 ug ml proteinase K prior to loading the gel SDS is added to reactions at 37 to facilitate trapping the enzyme in a cleavage complex Also if the reactions are heated cooled or treated with high salt prior to adding SDS the topo breakage and resealing equilibrium may be altered and br2. TopoGEN Copyright TopoGEN Inc 2012 All rights reserved User Manual Purified E coli DNA Gyrase and Relaxed DNA Kit plasmid based L Catalog Number TG2000G 1KIT 100 Reaction Set 50 ug DNA O Catalog Number TG2000G 3KIT 500 Reaction Set 250 ug DNA O Catalog Number TG2000G 5KIT 1000 Reaction Set 500 ug DNA Catalog Number TG2000G 7KIT 2000 Reaction Set 1 mg DNA Shipping and Storage of Reagents The kit is shipped on dry ice The DNAs should be stored at 4 C and the buffers stored at 20 C upon receipt Avoid frequent freeze thaw cycles with the plasmid as this may contribute to DNA breakage Store enzyme at 70 C We also recommend that the enzyme be aliquoted after the first thaw repeated rounds of freeze thaw may cause loss of activity the enzyme activity is stable for 1 3 days on ice TopoGEN Inc 108 Aces Alley Port Orange Florida 32128 USA Tel 614 451 5810 Fax 614 559 3932 Orders sales topogen com Support support topogen com Website www topogen com Product Application and Disclaimer This product is not licensed or approved for administration to humans or animals It may be used with experimental animals only The product is for in vitro research diagnostic studies only The product in non infectious and non hazardous to human health This information is based on present knowledge and does not constitute a guarantee for any specific product features and shall not establish a legally
3. are ideal for testing enzyme activity however the nicked open circular DNA product cannot be clearly identified in this gel system As noted above an EB containing gel 0 5 ug ml EB in gel and buffer will improve the resolution of cleavage products nicked open circular and linear DNA Be sure to destain with water for 15 min prior to photodocumenting your data In some cases depending on how the gel is run the topoisomer distribution can interfere with your ability to see the cleavage products however EB gels remove this complication IMPORTANT If unsure about whether to run an EB or Non EB gel we suggest that you run both Simply divide your reactions into equal parts and run two gels at the same time In this way you will be sure to see all reaction products and enhance your interpretation of the experiment With all markers in the gel system supercoiled relaxed linear for example you will obtain very clear and unambiguous results What are the running conditions in terms of time and voltage Run gels at 1 5 2 V cm measured between electrodes until the dye front has traveled about 80 After running non EB gels should be stained with EB 0 5 ug ml for 15 30 min and then destained in water or buffer for 15 min prior to photodocumentation EB gels are run in the presence of 0 5 ug ml in gel and running buffer then destained with water for 15 min prior to photodocumentation IMPORTANT Try not to run the gels overnight
4. d DNA result based on an agarose gel that lacks ethidium bromide These non EB gels are ideal for detecting gyrase activity as attested by the excellent resolution between supercoiled and relaxed DNA forms Gyrase Forms and stabilizes a positive gt DNA node one duplex over another Gyrase then Induces a transient DNA DS break on the underlying m duplex Gyrase then reseals the break on the front side of the complex One Negative Supercoil Introduced Relaxed pHOT1 Substrate Cycle repeats and fully supercoiled ou pHOT1 product results Relaxed Supercoiled Figure 1 The Gyrase reaction Substrate Product TopoGEN Inc www topogen com Protocol TG2000GKIT 2 Version E coli DNA Gyrase User Manual C DNA Gyrase Quality Control 1 A test for nuclease contamination was carried out by assaying for the formation of linear Kinetoplast DNA kKDNA and linear plasmid DNA Incubations of 1 ug of catenated kKDNA or supercoiled pUC19 DNA 4 hrs at 37 C in the presence of 10 mM MgClz were performed Linear DNA or breakdown products were not generated under these conditions 2 The A and B subunits are gt 95 pure based upon SDS PAGE and certified to be endonuclease free 3 Note with high input levels of DNA gyrase a small amount of linear DNA may be detected due to spontaneously ab
5. eaks can reseal Why is proteinase K required Drugs that trap topo DNA complexes will induce covalent complexes between DNA and protein topo and this protein must be removed degraded Failure to do so will prevent detection of the cleavage products If the reactions are heated cooled or treated with high salt prior to SDS the topo DNA breakage and resealing equilibrium may be altered and breaks can reseal Can you help us with data interpretation Yes we can definitely help The best way to proceed is to send us your data support topogen com with a full description of the experiment We will get back to you quickly with feedback Can you show us some real gel data and discuss the results Yes we can The results and a helpful discussion is shown in Fig 2 see legend Any further questions or comments please feel free to contact us support topogen com TopoGEN Inc www topogen com Protocol TG2000GKIT 7 Version E coli DNA Gyrase User Manual Gyrase RelDNA Gyrase Linear DNA Ciproflox Relaxed e Topoisomers lt Linear Linear DNA DNA Supercoiled DNA Figure 2 Gyrase Reaction Products Resolved on a Non EB Gel system Gyrase reactions were carried out in a final volume of 20 ul see above protocol Reactions were terminated with 1 SDS digested with proteinase K and extracted with CIA The Gel were run at 50v for 45 50 min and stained with EB non EB gel per pr
6. orted reactions This is normal D Dilution Buffer Dilution of purified DNA gyrase should be performed in 50 mM Tris Cl pH 7 5 100 mM NaCl 2 mM dithiothreitol 1 mM EDTA and 50 glycerol E Supercoiling Assay Conditions One unit of gyrase is typically incubated with 0 1 to 0 5 ug of relaxed plasmid DNA in a reaction volume of 20 30 ul for 1 hr at 37 C in assay buffer Assay buffer 1x recipe is shown below kit includes a 5x stock based on this formula 35 mM Tris Cl pH 7 5 24 mM KCI 4 mM MgCl 2 mM dithiothreitol 1 8 mM spermidine 1 mM ATP 6 5 glycerol 0 1 mg BSA ml F Relaxed DNA Quality Control Tests 1 Purity was evaluated spectrophotometrically using A260 A280 readings 2 Incubations with gyrase buffer alone at 37 C for 60 min did not result in formation of nicked or linear DNA species 3 Relaxation of supercoiled DNA was carried out using TopoGEN s purified DNA topoisomerase TG2005H RC1 Greater than 95 of the plasmid is relaxed under these conditions TopoGEN Inc www topogen com Protocol TG2000GKIT 3 Version E coli DNA Gyrase User Manual G Kit Contents Volumes are given for the TG2000G 1KIT size For the larger kits size multiply volumes as appropriate see page 1 for Kit sizes gt Relaxed pHOT1 DNA 50 ug total DNA concentration is specified on page 1 gt Supercoiled pHOT1 DNA 25 ul in gel loading buffer Load 2 ul as a marker gt 5x Gyrase assay buffer 600
7. otocols given above The data show the positions of relaxed DNA as well as gyrase reaction products supercoiled DNA The reaction with a fluoroquinolone ciprofloxacin yields a linear DNA cleavage product in the last lane on the right marked with a small triangle For full interpretation of gel data it is important to include relaxed DNA linear DNA markers as well as a control gyrase and gyrase drug as shown here TopoGEN Inc www topogen com Protocol TG2000GKIT 8 Version
8. reaction 20 ul order of addition is shown v v v Sterile Distilled H20 Vary as needed to bring volume up to 20 ul 5x Assay Buffer 4u pHOT1 Relaxed DNA 1 ul vary as needed The amount of relaxed DNA depends on sensitivity and detection methods With routine staining using ethidium bromide about 250 ng of DNA is usually sufficient however less may be used with more senstitive stains Gyrase 1 ul always add enzyme last on ice and transfer all tubes to heat block to initiate reaction sequence Procedure for a non EB gel analysis of DNA Gyrase Supercoiling 1 2 9 Incubate 30 60 min at 37 C Add 1 5 vol of stop buffer loading dye Add proteinase K to 50 ug ml digest for 10 30 min at 37 C optional step Add 20 ul of Chloroform isoamyl alcohol 24 1 mixture vortex briefly withdraw blue aqueous phase Load blue phase onto a 1 agarose 50x TAE buffer 242 g Tris base 57 1 ml glacial acetic acid 100 mI 0 5M EDTA Electrophorese until dye travels 60 75 down the gel Stain with 0 5 ug ml ethidium bromide for 30 min caution EB is a mutagen WEAR GLOVES Destain distilled water for 10 30 min room temperature handle gel with gloves Photograph using UV transilluminator J Analysis of reaction products by electrophoresis For each gel run a negative control relaxed DNA lacking gyrase The negative control should look very different from the gyrase reaction products which will be fa
9. ster migrating supercoiled DNA forms see Fig 2 TopoGEN Inc www topogen com Protocol TG2000GKIT 5 Version E coli DNA Gyrase User Manual J Frequently asked questions What are the critical controls to allow me to clearly identify drug that is affecting DNA gyrase using this assay Marker DNAs supercoiled linear DNAs are extremely important Be sure to run a positive DRUG control like a fluoroquinolone to demonstrate good cleavage activity You should see an increase in linear DNA Include a negative control either no drug or a topo Il drug such as etoposide VP16 or etoposide is not supplied but we have it available Be sure to check solvent effects Solvents like DMSO or methanol are used to dissolve some test drugs Test with a control reaction lacking drug but with solvent e g 1 DMSO What kind of agarose should buy Any nuclease free agarose of reasonable quality from any number of sources can be used Sigma Aldrich works What is the best gel buffer to use Agarose gels 1 and running buffers can be any standard nondenaturing electrophoresis buffer example to prepare a 50x of TAE Gel Buffer 242 g Tris base 57 1 ml glacial acetic acid and 100 ml of 0 5 M EDTA Dilute to 1x to use for gel separations Be sure that the gel also has 1x TAE buffer Should I run EB or Non EB gels and how do I run the EB gels In general 1 gels in the absence of Ethidium Bromide EB can be used these gels
10. ul 1x buffer contains recipe is given above gt Dilution buffer 600 ul Dilute gyrase with this buffer Recipe for dilution buffer given above gt 5x Stop Buffer gel loading dye 600 ul 5x buffer is 5 Sarkosyl 0 125 bromophenol blue 25 glycerol gt Purified DNA gyrase unit definition given on page 1 H Protocol for a typical reaction mixture final volume of 20 ul Protocol Summary Reaction volumes should be 20 30 ul final volume generally limited by volume that can be loaded onto the gel Reactions are assembled in microfuge tubes with water buffer and substrate pHOT1 relaxed DNA The gyrase should be added last and the reactions incubated at 37 C for 15 60 min or longer as appropriate then terminated with the stop load buffer proteinase K digested extracted and loaded onto an agarose gel note DO NOT add ethidium bromide EB to gel or gel buffer during electrophoresis use EB to stain the gel AFTER electrophoretic separation In some cases you may wish to run an EB gel 0 5ug ml EB IN GEL AND BUFFER in order to resolve nicked and linear DNA from circular substrate relaxed pHOT1 and supercoiled DNA products If you are unsure which to run we recommend that you try running both gel systems and divide your samples into two parts Usually there is sufficient DNA in the reaction to run two gel systems TopoGEN Inc www topogen com Protocol TG2000GKIT 4 Version E coli DNA Gyrase User Manual I Sample
11. valid contractual relationship TopoGEN Inc shall not be held liable for product failure due to mishandling and incorrect storage by end user TopoGEN s liability is limited to credit or product replacement E coli DNA Gyrase User Manual l Introduction A Summary This kit contains purified bacterial E coli DNA Gyrase purified to homogeneity based on SDS PAGE DNA Gyrase is prepared from overexpressing strains and is supplied as purified holoenzyme in an A2B2 complex The enzyme is supplied at the unit concentration given on the above quality control data provided with the kit The DNA Gyrase is stored in a stabilization buffer 50 mM Tris Cl pH 7 5 100 mM KCI 2 mM dithiotreitol 1 mM EDTA 50 glycerol Also included is substrate DNA supplied in TE 10 mM Tris HCI 1 mM EDTA pH 7 5 DNA concentration of the relaxed DNA is provided on the label of the tube The DNA Gyrase Assay Kit is based on supercoiling action on a relaxed plasmid DNA substrate provided pHOT1 which is a derivative of pBR322 and roughly 2 7 KB One unit of gyrase will supercoil 500 ng 0 5ug in 1 hr at 37 C under conditions defined below The reaction mechanism is describe below in Fig 1 The enzyme introduces negative supercoils into a relaxed pHOT1 plasmid substrate via a sign inversion model An energy co factor ATP is required for the complete supercoiling reaction The inset gel image in Fig 1 represents a typical substrate and product supercoile
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