text:
The use of a gel produced by polymerization of a dextran derivative which exhibits groups that contain an alkene structure, for separating nucleic acid by electrophoresis.
1. In a method for separating nucleic acids by the electrophoresis, wherein a gel is utilized as an electrophoresis medium and an electric field is applied across the surface of the gel to separate the nucleic acids, the improvement comprising using, as the gel, a gel prepared by polymerization of a dextran derivative having groups that contain an alkene structure.
2. The method according to claim 1, wherein said groups have the structure
R is H, hydrocarbon group, F, Cl, Br or CN, and
the free valance binds to an hydroxylic oxygen in dextran, and
B is a straight, branched or cyclic hydrocarbon bridge which is optionally broken by ether oxygen and/or substituted with one or more alcoholic hydroxyl groups.
3. The method according to claim 2, wherein R is hydrogen.
4. The method according to claim 2, wherein B is �CH2� or �CH2�O�CH2�CH(OH)�CH2�.
5. The method according to claim 2, wherein R is CH3.
6. The method according to claim 1, wherein the nucleic acid is DNA.
7. The method according to claim 1, wherein the nucleic acid is single-stranded.
8. The method according to claim 1, wherein the nucleic acid is double-stranded.
9. The method according to claim 1, wherein the nucleic acid has less than 20,000 bases if single-stranded or less than 20,000 base pairs if double-stranded.
10. The method according to claim 9, wherein electrophoresis is carried out at a temperature at which the nucleic acid exists in single-stranded form, preferably >50� C. or in the presence of a denaturing agent which causes the nucleic acid to exist in single-stranded form at pH 7-10 or at elevated pH 10-13.
11. The method according to claim 1, wherein said gel is produced by polymerization of a dextran derivative exhibiting groups having an alkene structure, and wherein said gel includes a stacking zone of less dense gel structure and a separation zone of denser gel structure.
12. The method according to claim 1, wherein said gel is produced by polymerization of a dextran derivative exhibiting groups having an alkene structure, and wherein said gel is carried by a supporting plastic material.
13. The method according to claim 12, wherein said supporting plastic material is an intermediate film that enhances adhesiveness and stability.
14. The method according to claim 1, wherein said gel is produced by polymerization of a dextran derivative exhibiting groups having an alkene structure, and wherein said gel is cast in channels or in rectangular moulds.
15. A gel produced by polymerization of a dextran derivative exhibiting groups having an alkene structure comprising:
R is H, a hydrocarbon group, F, Cl, Br or CN, and
the free valance binds to an hydroxylic oxygen in dextran, and
B is a straight, branched or cyclic hydrocarbon bridge which is optionally broken by ether oxygen and/or substituted with one or more alcoholic hydroxyl groups
said gel being hydrated with a buffer giving a pH value in the range of 10-13.
16. A gel produced by polymerization of a dextran derivative exhibiting groups having an alkene structure comprising:
R is H, a hydrocarbon group, F, Cl, Br or CN, and
the free valance binds to an hydroxylic oxygen in dextran, and
B is a straight, branched or cyclic hydrocarbon bridge which is optionally broken by ether oxygen and/or substituted with one or more alcoholic hydroxyl groups
said gel being hydrated with a buffer set to a pH value in the range of 7-10 and containing a denaturing agent which dissociates double-stranded nucleic acid.
The invention relates to the electrophoresis of single-stranded or double-strand nucleic acid (ss and ds respectively), primarily polynucleotides that consist of sequences of 5-20,000 nucleotides (alternatively base pairs).
It is known that gels based, inter alia, on agarose, polyacrylamides, bis-acrylamide, etc., can be used in the electrophoresis of nucleic acid.
The gels have normally been cast or molded in the presence of an electrophoresis buffer.
In the electrophoretic analysis of single-stranded DNA (sequencing) separation is normally effected in the presence of urea in order to keep the strands dissociated (Maniatis et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989) New York, U.S.A.). Consequently, gels have been cast together with urea at a pH of about 8 in direct association with their use. Efforts to incorporate buffers of a higher pH value have also been made (Smith et al, Amer. Biotechnol. 8 (1990), 48-54).
Problems Associated with Hitherto Known Techniques, and Desirable Properties of Relevant Gels
Agarose gels that possess good sieving properties for the DNA-sequencing of samples are difficult to produce. Agarose also has the drawback that the gels readily become opaque and therefore are difficult to use in conjunction with optical detection. Drawbacks with polyacrylamide in electrophoresis is that the monomers (acrylamide and bisacrylamide) are toxic, and consequently great care must be observed when handling the monomers, and also that acrylates and acrylamides are hydrolysis sensitive. In the case of prefabricated gels that contain urea, the urea is also readily hydrolyzed.
As an alternative of subjecting single-stranded nucleic acid to electrophoresis in the presence of a denaturing agent, such as urea, electrophoresis may be carried out at an elevated pH (normally about pH 11) and/or at an elevated temperature (about 80-90� C.). Both alternatives result in the hydrolysis of polyacrylamide.
There is a strong general desire to find novel gel materials that can be used in the electrophoresis of nucleic acid. Objectives with regard to novel gel materials are that they shall:
1. Enable separation of single-stranded and/or double-stranded nucleic acid, primarily DNA, with a resolution that is comparable with or superior to commercial polyacrylamide gel (=PAA).
2. Enable the fabrication of gradient gels and gels that have different zones.
3. Provide low electro-endosmosis, preferably equal to or better than PAA.
4. Be stable in the pH range relevant to electrophoresis, normally 4-13.
5. Tolerate urea 8 M and other denaturing agents and conventional staining techniques for nucleic acid (e.g. silver staining and ethidium bromide staining).
6. Possess good optical properties, i.e. high light transmission 280-800 nm and non-disturbing (low) fluorescence, particularly at 480, 590 and 630 nm.
7. Be non-toxic.
The use of dextran derivative gels for protein electrophoresis was proposed about twenty years ago (S�derberg, L., U.S. Pat, No. 4,094,832). The gels were produced by free radical polymerization of a dextran derivative containing vinyl groups chosen from
where �A� is �CH2� or �O� and R is H, CH3, F, Cl, Br or CN. Polymerization could optionally be effected in the presence of low molecular vinyl compounds. Protein electrophoresis was the stated application. The advantages obtained were said to be that the gels were inert against biological substances, were stronger, could be cast with an incorporated electrophoresis buffer, could be cast or molded in the form of easily handled plates having tailor-made properties in accordance with the choice of vinyl substituents, charged substituents and copolymers, etc.
In conjunction with the attempt to produce electrophoresis gels that would give improved resolution of low molecular compounds, low �smiling�, and improved optical properties, there have been proposed gels of polyhydroxy polymers cross-linked with a reagent that provides ether bridges with OH groups in the polymer (Kozulic, B., U.S. Pat. No. 5,371,208; see in particular column 11). The function of the gels has been shown for electrophoresis of DNA digested with the aid of restriction enzymes. Allyl agarose (Example 1) and dextran (Examples 14 and 16) can be mentioned from among a large number of examples.
A copolymer between acrylamide and allyl substituted agarose (Nochumsson, S., EP 87,995) has been proposed as a gel for electrophoresis, since such gels adhere well to conventional carrier or substrate materials.
The object of the invention is to find novel gels for electrophoresis, which are suited for the separation of nucleic acid and which have improved properties with regard to the aforesaid objectives.
The invention
It has now been found that electrophoresis gels polymerized from a dextran derivative that includes groups having an alkene structure (>C═C<) are suited for electrophoretic separation of both single-stranded and double-stranded nucleic acids. The gels have improved properties with regard to several of the features mentioned in the aforegoing, such as chemical stability at high pH levels, optical clarity, are non-toxic and have a long shelf life. The polymer may be in the form of a copolymer with low molecular vinyl monomers. The gels concerned have earlier been defined in principle (S�derberg, L., U.S. Pat. No. 4,094,832).
The main aspect of the invention resides in a method for the gel electrophoretic separation of nucleic acid. The main feature of the method resides in the use of a gel which is a free radical-polymer of the same type as that used by S�derberg (U.S. Pat. No. 4,094,832). The method includes the steps typical in the gel electrophoresis of nucleic acid, i.e. the sample is placed on the gel and a voltage applied. When wishing to carry out electrophoresis on single-stranded DNA, the process is effected in the presence of a denaturing agent which dissociates double-stranded nucleic acid. See below. Alternatively, the electrophoresis can be carried out at elevated temperatures (e.g. temperatures above 50� C., a denaturing agent may be present when necessary).
Preferred groups that include alkene structures have the formula
where R may be H, F, Cl, Br or CN or a straight, branched or cyclic hydrocarbon group which may be saturated or unsaturated or contain an aromatic structure. Among saturated hydrocarbon groups, for instance groups according to the formula CnH2n+1 (n is an integer) can be mentioned those that contain ten or fewer carbon atoms (n integer≦10). The most preferred groups R are hydrogen (H) and lower alkyl groups having 1-4 carbon atoms, particular methyl (CH3). The hydrogen atoms in H2C═ may be substituted with halogen, particularly Cl or F. The hydrocarbon chain in a group R may be broken or one or more of the carbons may be substituted with one or more oxygen atoms in the same manner as the bridge B (see below).
The bridge B binds directly to an oxygen atom in a hydroxyl group of dextran. The bridge B is a straight, branched or cyclic hydrocarbon bridge which may be saturated or unsaturated and is optionally broken by ether oxygen and/or has one or more carbon atoms that are substituted with one alcoholic hydroxyl group. The bridge B may include aromatic structures, such as phenylene (as cyclic structures; �C6H4�). The bridge B may alternatively be a single bond. At most, one oxygen atom is directly bound to one and the same carbon atom. The length of the bridge is normally less than 1-15 atoms, including any oxygen atoms. Unsubstituted hydrocarbon bridges are for instance �(CH2)n�, where n is an integer chosen from 1-10, for instance 2, 3 or 4. Phenylene, cyclohexandiyl, etc., are examples of cyclic structures that can be included in the bridge.
Specific examples of the group H2C═CR�B� allyl, 3-allyloxy-2-hydroxy-propyl, 3-(styryl-4-oxy)-2-hydroxypropyl, 1-methylallyl, 1-chloro-allyl and non-substituted vinyl, etc. See S�derberg, L. (U.S. Pat. No. 4,094,832) for further examples.
The substitution degree of the dextran derivative with respect to alkene-containing groups is suitably 0.05-1.9, preferably 0.5-1.2, mmol/g dry dextran derivative (=1-30% and 5-25% respectively).
The molecular weight of the dextran derivative may vary within wide limits. Starting from non-substituted dextran, the mean molecular weight (Mw) will preferably lie in the range of 10,000-10,000,000 Da, particularly 70,000-5,000,000 Da.
By suitable combinations of polymerization conditions (substitution degree, initiator, dextran concentration, the molecular weight of dextran, etc.) gels that have different properties can be produced for the electrophoresis of nucleic acid.
The dextran derivative is produced in a known manner (see, for instance, S�derberg, L., U.S. Pat. No. 4,094,832).
The low molecular vinyl compound (normally a monovinyl compound or divinyl compound) with which copolymerization may be carried out may be an acrylamide or a methacrylamide which may be substituted at the nitrogen atom or at one of the double-bonded carbon atoms. Alternatively, there may be used other low molecular vinyl compounds that have equivalent stability properties against hydrolysis in the relevant pH ranges and temperature ranges. The vinyl compound will preferably contain at most twenty carbon atoms, preferably at most ten carbon atoms. C.F. S�derberg, L., U.S. Pat. No. 4,094,932.
The polymerization conditions are those typical for the polymerization of alkene groups (electron-radiation or γ-radiation, oxidative radical polymerization, etc.), preferably free radical polymerization being chosen in this regard. UV-initiators of, e.g., a benzophenone or a benzoyl derivative, and chemical initiators such as persulphates, peroxides and azobisisonitrile can be used. Accelerators are used when necessary. The quantity of dextran derivative used normally corresponds to 20-100% (w/w) calculated on the total amount of polymerizable reactants.
Dextran gels can be cast with a buffer system incorporated in the polymerization solution. For instance, there can be produced prefabricated gels which include buffer components that provide a pH within the relevant pH range, e.g. pH 7-10 or 10-13. The buffering components will normally have pKa values within the relevant ranges. In accordance with the aforegoing, denaturing agents that dissociate double-stranded nucleic acid, primarily urea and formamide may be incorporated in respect of the lower range. See Levine et al, Biochemistry 2(1) (1963), 168-175 (which is herewith incorporated in the present document by reference). With regard to the upper range, suitable buffer components are bases that are stronger than ammonia, for instance borates, soluble hydroxides (OH−), carbonates (CO3 2 �), phosphates (PO43�) and certain primary and secondary amines, of which 2-amino-2-methyl-1-propanol can be mentioned among others. The buffer components will preferably not be volatile, implying that their boiling points will normally be above 120� C. Typical electrophoresis buffer systems are described in U.S. Pat. No. 5,055,517 (column 15-16) (which is herewith incorporated by reference).
The gels can be cast so as to contain different zones, e.g. a less dense stacking zone and a denser separation zone. The gels may also be cast as gradient gels and in different geometries, such as in rectangular cassettes, in channels of different cross-sectional shapes, including capillaries.
Gels of the type concerned are often relatively weak from a mechanical aspect. When the gels are to be treated as a �slab� gel, the gels will preferably be cast on a carrier or substrate in a known manner, normally a polymer film (e.g. GelBond� PAG (FMC, USA)).
According to one particular aspect of the invention, an electrophoresis gel is fabricated from a dextran derivative polymer in accordance with the aforegoing. This aspect of the invention is characterized in that the gel includes buffer components and denaturing agents according to the aforegoing.
For further information relating to conditions for polymerization and the production of dextran derivatives, the reader is referred to the aforementioned patent publications (incorporated by reference) and also to textbooks on the subject.
Allyl dextran solutions (Mw 2,000,000 g/mol, allyl content of 1.2-1.5 mmol/g dry substance (CH2═CH�CH2� being substituted at an OH of dextran), were prepared by dissolving a given quantity of allyl dextran in a volume of buffer (TRIS-HCl 0.375 M pH 8.8), which was slightly less than desired. This is because the volume of the buffer increases when dextran dissolves. After all allyl dextran had dissolved, the volume was adjusted to the desired volume with buffer. Urea was added in certain instances.
A. Gel Containing Urea, pH about 8.2
A solution containing 14% w/v allyl dextran in TRIS-HCl (0.375 M, pH 8.8) and urea (7 M) was prepared. 250 μl ammonium persulphate solution (0.1 g/ml in TRIS-HCl 0.375, pH 8.8) was stirred into 50 ml of the solution of allyldextran. The solution was de-aerated for about 3-5 minutes with the aid of a vacuum pump. 25 μl of an accelerator (TEMED=N,N,N′,N′-tetramethylethylenediamine) while carefully stirring so as to avoid contamination with air. The mixture was allowed to stand for roughly 5 minutes, until all air bubbles had disappeared. The solution was then sucked into a syringe (30ml) and a cassette (short 15 cm) for ALFexpress DNA Sequencer (Pharmacia Biotech AB, Uppsala, Sweden) was filled with a gel by capillary suction of the solution. It took roughly 5 minutes to fill the cassette and roughly 30-45 minutes to polymerize the solution, counting from the time at which TEMED was added.
The gel was evaluated electrophoretically in an ALfexpress DNA Sequencer, by separating single-stranded DNA (ssDNA). The samples separated were fragment samples comprising a 50-base pair ladder 50-500 bp and a sequence sample M13. Both samples were labelled or marked with Cy-5 and had been obtained from Pharmacia Biotech AB, Reagents Division (Milwaukee, U.S.A.). The samples were prepared in accordance with standard methods for processing on ALFexpress with polyacrylamide gel. The tank buffer was TRIS-borate-EDTA (0.1M, 0.08 M, 1 mM, pH 8.2). The gel was processed with the following parameters: U=1000 V, I=60 mA, P=15 W, T=55� C. The effect was limiting. The gel was pre-processed with these parameters for 50 minutes prior to adding the samples.
B. Gel Without Urea, pH about 11
A solution containing 13% w/v allyl dextran in 2-amino-2-methyl-1-propanol (100 mM, pH 11.2) was prepared. 500 μl of an ammonium persulphate solution (0.1 g/ml in solution of 2-amino-2-methyl-1-propanol (100 mM) was stirred into 50 ml of the solution of allyl dextran. The solution was de-aerated with the aid of a vacuum pump over a period of about 3-5 minutes. 50 μl TEMED were then added, while carefully stirring the system. The solution was then treated in the same manner as in the preceding experiment prior to casting the gel in a cassette (short 15 cm) for an ALFexpress DNA Sequencer.
The gel was evaluated with the same samples and in the same way as in the preceding experiment. The tank buffer was 100 mM 2-amino-2-methyl-1-propanol, pH 11.2.
Results and Discussions
Gel Casting for ALFexpress DNA Sequencer
The initiator system ammonium persulphate-TEMED functioned well for allyl dextran. The time taken to fill a gel cassette with reaction solution was slightly longer than in the case of a conventional acrylamide solution. It was possible to adapt the induction time so as to enable the solution to be de-aerated and the gel cassette filled prior to commencement of the polymerization process. On a molar basis, the amount of initiator used when urea was present was the same as the amounts normally used for the preparation of acrylamide-urea gels for ALFexpress DNA Sequencers. The double amounts of the initiator and accelerator were required to obtain polymerization within the same time frames without urea but with 2-amino-2-methyl-1-propanol in the buffer. This is probably because polymerization is inhibited at a high pH and when amines are present, therewith requiring higher concentrations of initiator plus accelerator in order to start polymerization.
Optical Quality
The gels that had been cast with the initiator system ammonium persulphate plus TEMED exhibited sufficiently good optical clarity to enable laser-induced florescence to be used for detection purposes (e.g. ALFexpress DNA Sequencer). The solutions from which the first gels were cast were not de-aerated prior to casting. This resulted in tendency towards less clear gels. It is probable that small air bubbles cause the light to be spread and therewith result in less clear gels, which in turn makes laser detection more difficult to achieve.
Electrophoresis, Gel Containing Urea, pH 8.2
The system was pre-run for 50 minutes, since the selected buffer system was discontinuous (TRIS-HCl in the gel and TRIS-borate-EDTA in the tank). Without pre-running, there is a danger that low molecular fragments will stack behind the chloride-ion front. In the case of the fragment containing sample, the primer peak arrives after about 20 minutes, followed by the ten fragments. Separation was of the same quality as that achieved with polyacrylamide gel. Up to about 240 bases were separated for the sequence sample. The peaks then became wider and it was impossible to read a sequence. Of the 200 first bases, 16 were wrong. The faults, with the exception of one, were all deletions, i.e. where there should have been one or more of the same bases in sequence one less base was identified, for instance one of two or three of four. This deviation may have been because the algorithm used was not adapted to the gel. The tests showed that it is possible to separate ssDNA on an allyl dextran gel that is polymerized with the system ammonium persulphate plus TEMED, and that detection can be effected with laser-induced fluorescence.
Electrophoresis, Gel in the Absence of Urea, pH 11.2
The gel was run with a continuous system using the same buffer in tank and gel. In the case of the fragment sample, the primer peak arrived after little more than 20 minutes. The first fragment (50 and 100 bp) gave no distinct peaks (rounded). No explanation could be found for this. On the other hand, the subsequent fragments (150-500 bp) were very distinct and separation appeared normal. The total separation time was roughly the same as that for corresponding allyl dextran gel including urea (pH 8.2). No sequencing could be obtained in the case of the sequence sample (M13). The primer peaks could be seen, but then no more than noise. The experiment was repeated, but with the same result. According to information from the sample supplier, the fluorophore in the labelled sample had limited stability at high pH values and for certain amines. This may be one explanation for the �no result� result for the sequence sample. Marking or labelling of the primer and of the fragment sample was much stronger, which may be the reason why these could be shown. The obtained results show that gel containing no urea and having a high pH is able to separate single stranded fragment samples with approximately the same resolution as for a gel that contains urea and has a lower pH. More study is required to establish why the sequence sample could not be seen.
120 μl of ammonium persulphate solution (0.1 g/ml in TRIS-HCl 0.375, pH 8.8) were stirred into a solution of 17% w/v allyl dextran in TRIS-HCl (0.375, pH 8.8). The solution was deaerated for about 5 minutes with the aid of a vacuum pump. 15 μl TEMED=N,N,N′,N′-tetramethylethylenediamine were then carefully stirred into the solution. The solution was allowed to stand for about 5 minutes until all air bubbles had disappeared. The solution was then sucked into a syringe and delivered to a gel cassette consisting of a glass plate and an aluminium oxide plate. The gel had a size of 8.5�9 cm. A film (GelBond�, FMC, USA) of the same size lay against the aluminium oxide sheet. The film functioned as a carrier, because the allyl dextran gels were too weak mechanically to be handled on their own. The gel was cast in the absence of stack zones and with combs to create sharply defined wells for sample deposition. The polymerization process commenced about 45 minutes after adding TEMED. The gel was allowed to stand for one hour before being processed.
The gel was evaluated in Mighty Small SE 260 (Hoefer Scientific Instruments, California, U.S.A.) with the fragment samples Hinc and Hae (Hinc II DIGEST FX-174-RF (13 fragments from 1057-79 base pairs) and Hae III DIGEST FX-174-RF (11 fragments from 1353 to 72 base pairs) (Pharmacia Biotech AB, Uppsala, Sweden). The tank buffer used was TRIS-glycine (0.025 M, 0.192 M, pH 8.3). The gel was run for about 12 minutes with I=15 mA prior to charging the samples. The voltage varied during this period from 60 V to 110 V. The samples were then delivered to the gel and separation was effected with I=25 mA. The gel was processed to the front with bromo-phenol blue was about 2 cm from the gel edge. This took about 50 minutes and the voltage changed from 190 V to 400 V and the power from 5 W to 10 W. The gel was developed by silver staining. The time for developing the gel was about 30 minutes longer than the time for developping a polyacrylamide gel. The surface of the gel became �crackled� in the fixing bath (solution containing 10% acetic acid) in the development process. This may be due to surface contraction of the gel. This crackling made it difficult to interpret the obtained bands. All bands were not included, although the separation of dsDNA fragments was distinct.
Gels were produced in separate tests with the aid of an UV-initiator (Irgacure� 184 (1-hydroxy-cyclohexyl phenyl ketone) molecular weight 204, Ciba-Geigy) and the same allyl dextran derivative as that defined above. Preparation was found to be sensitive, due to the solubility properties of the initiator. The gels obtained by this method were fully in class with those obtained by chemical initiation (ammonium persulphate plus TEMED).
Summary: Other Properties of the Synthesized Gels
No disturbing electroendosmosis could be observed with the gels. Turbidity has, as a rule, been comparable with that obtained with conventional polyacrylamide gels. The intrinsic fluorescence values measured at 630 nm showed that background fluorescence was acceptable. Backgrounds of 3% were measured at 630 nm, which is slightly higher than the values obtained with polyacrylamide gels. This is in no way disturbing, however. Adhesion with regard to relevant carrier materials was good. With regard to GelBond Pag, the gel adhered both to the hydrophilic and the hydrophobic side. The finished gel remained seated during treatment with those solutions applicable for silver staining. The mechanical properties of the gels render them weaker than polyacrylamide gels. They are readily torn if handled in the absence of a carrier substrate. No problem with regard to swelling of the gels was encountered during the electrophoresis.
Experimental Support Obtained During the Priority Year
During the priority year Swedish patent application 9600612-7 has been filed. It deals with polyhydroxy polymers carrying styryl ether groups. SE 9600612-7 is hereby incorporated by reference.
The application gives the manufacture of styryl glycidyl ether by first synthesizing 4-(2,3-epoxypropoxy)benzaldehyde from 4-hydroxy benzaldehyde and epichlorohydrin in the presence of NaOH, tetra-n-butyl ammonium iodide and dimethylsulfoxide. 4-(2,3-epoxypropoxy)benzaldehyde was then added to a dry tetrahydrofuran solution (0� C., argon atmosphere) containing suspended methyl-triphosphonium bromide, catalytic amounts of 18-crown-6-ether and potassium t-butoxide. From this mixture styryl glycidyl ether was then obtained in a yield of 70%.
Styryl glycidyl ether was then reacted in various ways with dextran (Mw 40,000 Da or 500,000 Da). Various ratios styryl glycidyl ether to dextran, various solvents (pure water, pure DMSO), various mixtures of solvents (water, DMSO, toluene). various amounts of bases (NaOH and where appropriate sodium dimsyl) and in some instances also a phase transfer catalyst were used. Degrees of substitution ranging from 0 up to 50% were accomplished.
A styryl ether dextran so obtained (degree of substitution 2.8%) dissolved to a concentration of 15% (w/v) in urea (7M) containing Tris-HCl buffer (0.375 M, pH 8.8) was polymerized to a gel with a radical initiator. The utility of the gel was then illustrated by subjecting a sample containing double-stranded DNA to electrophoresis on the gel.
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FOXBORO — For all the talk about the Miami's vaunted Wildcat offense, it was special teams that sunk the Jets last week against the Dolphins, specifically Ted Ginn Jr.
A week after being demoted from his usual role of wide receiver to full-time special teamer, Ginn returned a pair of kicks for touchdowns in Miami's win over New York at the Meadowlands, one going for 100 yards and the second going for 101. He became the eighth player in NFL history to have two kickoff returns for touchdowns in the same game, and the first NFL player to run back two kickoffs for touchdowns in the same quarter since Green Bay's Travis Williams did it in 1967. Ginn ended up with 299 returns yards on the day.
The performance sparked hyperbole from Jets special teams coach Mike Westhoff.
"The worst. Worst ever for me, ever," Westhoff told reporters who asked about Ginn on Monday. "Absolute worst. We gave them the game. … It won't go away until they lower me in the grave."
Patriots special teams captain Sam Aiken watched Ginn's performance, and was suitably impressed.
"No matter what angle you take, you're not catching him," Aiken said when asked what he took from Ginn's performance. "That's why they draft guys like that, to make big plays like that. You take those two returns away, it's a different game. You have guys back there you know can make big plays."
Ginn is just the latest big-time kick returner New England will face this season. Buffalo's Leodis McKelvin was one of the best in the league in 2008, and racked up nearly 100 yards on four returns in the season opener against the Patriots. New York's Leon Washington was in the top five in most major categories last season, and gashed New England with a game-changing 43-yard return in Week 2. The Patriots were lucky to duck Tampa Bay's Clifton Smith, a kick returner in the top five in most major return categories this season.
No such luck this week, as Ginn appears ready to play. Thanks in large part to what he did last week, the Ohio State product leads the league in average yards per return (34.9) and touchdowns (two).
Don't expect Westhoff-style hyperbole from Patriots coach Bill Belichick, but Belichick said Ginn has every right to be mentioned in the same class as McKelvin, Washington or Smith.
"We've seen top returners every week, and this is another one," Belichick said of Ginn. "I don't know that we or any team in the league has more than one or two, if that, people that could run with Ted Ginn.
"Leverage, tackling and discipline in our lanes and recognizing blocking schemes and doing a good job of tackling — all that is going to be crucial against Miami this week."
There's no doubt he has the speed, as well as the rest of the obvious physical characteristics needed to succeed as a return man. But according to Aiken, it's not just the physical skill set that makes Ginn special. Aiken said Ginn has the rare gift of having what he calls "the total package" in a kick returner — vision and instinct.
"It's the total package," Aiken said of Ginn. "To be a good kick returner, you have to have vision. You have some guys who are fast, but can't react. You have some guys who are fast and can react because they can see the opening. They can see where the defender is coming from.
"It's all about vision and instinct."
The Patriots are in the middle of the pack when it comes to kick coverage: they are no higher than 11th, no lower than 20th in every major category. When they have had success in kick coverage, it's because they've stuck to the fundamentals, something they've done lately. As a result, the average yardage allowed on each kickoff has decreased steadily, from 31.8 yards allowed per return in Week 2 against the Jets to 17.7 yards per return in a Week 7 win over Tampa Bay.
"Keep doing what we've been doing," Aiken said. "Stay in focus. Maintain your lanes. Keep doing what we've been doing."
Hall of Famer Michael Irvin checks in with the Dale and Holley show and offers up some perspective on the Seahawks locker room situation, and whether the supposed discord amongst other players and quarterback Russell Wilson is part of the reason why the Seahawks have struggled on the field of late.
Devin McCourty discusses the dangerous Bears offense.
Adam Schefter breaks down the Patriots and Darrelle Revis, Russel Wilson and the Seahawks, and Jay Cutler and the Bears
Jackie MacMullan joins MFB for the first time this season. She discusses the rejection of NBA lottery reform, Rajon Rondo, and the Celtics chances this season.
Kirk Minihane joins the We Hate Dwyane Wade Show to preview the upcoming Celtics season. Ben and Sam also discuss the Rajon Rondo/Jeff Green trade rumors and dabble in the classic NBA analytics debate.
Joe Castiglione does his annual wrap-up of the season by reading the poem by Bart Giamatti.
Mut and Rob discuss the "kids" and the variety of young bodies the Red Sox called upon this season. Specifically, they look at Bogaerts and Bradley, Jr. and where things went wrong. They give their opinions on which one will right the ship next season.
Description: Red Sox farm director Ben Crockett looks back at the 2014 Red Sox prospect season by answering listener questions about a number of Red Sox prospects as well as broader issues and themes in the farm system.
Joe McDonald joins the guys to talk to about the Chara injury and how the Bruins will move forward without him.
Pierre McGuire joins the guys to talk about the Bruins, the Johnny Boychuck trade, and the rest of the NHL
We talk pucks with Jack Edwards of NESN for his take on an impressive win over the San Jose Sharks and the state of the B's in general.
Rodney discussed Russell Wilson and the Seahawks.
The top stories of the day as recounted by Kirk Minihane.
The guys discussed Freeman's story on Russell Wilson.
Devin McCourty discusses the dangerous Bears offense.
Adam Schefter breaks down the Patriots and Darrelle Revis, Russel Wilson and the Seahawks, and Jay Cutler and the Bears
Hall of Famer Michael Irvin checks in with the Dale and Holley show and offers up some perspective on the Seahawks locker room situation, and whether the supposed discord amongst other players and quarterback Russell Wilson is part of the reason why the Seahawks have struggled on the field of late.
We discuss another impressive win by Peyton Manning and the Broncos and think long term for the 2014 season about whether or not the Patriots will have enough firepower to beat the Broncos in crunch time: the playoffs.
We tackle four topics that pit Boston against Chicago in one form or another.
Mikey, Villani and Lenny are talking about the 2004 Red Sox on the anniversary of Game 4 of the ALCS with Dave Roberts steal abd Bill Mueller driving him in and what it was like to witness.
Mikey, Mut and Lenny are talking about the Patriots and how they have stacked up through the years and what got them to the three championships and five Super Bowl appearances in the Brady-Belichick era.
Mikey and Mut take a call from someone who wants to criticize Belichick the GM and his history of drafting players and making moves in the draft.
AJ Soares joins the show to talk about the Revs run to the playoffs, how Jermaine Jones has impacted the team, and should Lee Nguyen for MVP.
The Dennis & Callahan Show producers fight back.
Rodney discussed Russell Wilson and the Seahawks.More from this show
Sausage and Lumpy, the producers for the D&C show, recorded a podcast yesterday. They left A LOT of stuff on the cutting room floor. Luckily, MFB picked it all up and played the unedited version during the midday show. Kirk joined the show to discuss how hard it is working with Callahan and his two producers.More from this show
The guys opened the show discussing the disgusting weather.More from this show
The guys discussed hockey's place on the American sports landscape.More from this show
We discuss the breaking news that Darrelle Revis was sent home yesterday for being late to scheduled team activities. Was it the right move by Bill Belichick? We discuss.More from this show

subset: commoncrawl


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Friday, April 30, 2010
- BP Oil Spill Hits Louisiana Coastline
- Thousands Gather for March on Wall St.
- Goldman Sachs Faces Criminal Probe
- Protests, Lawsuits Challenge Arizona Immigration Law
- Pentagon: Afghan Violence Up Nearly 90%
- NATO Forces Kill Relative of Afghan Lawmaker
- UN Closes Kandahar Mission
- Gitmo Prisoner Skips Evidentiary Hearing
- House Approves Puerto Rico Referendum
- 4 Prisoners in Holy Land Case Moved to Secretive, Restrictive CMUs
"You Got Bailed Out, We Got Sold Out"–Thousands Protest on Wall Street
Thousands of people turned out for a protest on Wall Street Thursday to denounce the taxpayer-funded bailout and the role of large financial firms in the nation's economic crisis. A coalition of union and community groups organized the march as the Senate opened debate on a measure to overhaul financial regulation. [includes rush transcript]
Spewing 5,000 Barrels of Oil a Day, BP Spill Hits Louisiana Coastline
The massive BP oil well leak in the Gulf of Mexico has reached the Louisiana coastline as fears grow of a worse disaster than the 1989 Exxon Valdez oil spill. 5,000 barrels of oil a day continue to spew into the water beneath the site of the Deepwater Horizon rig that exploded and sank last week. President Obama said BP is ultimately responsible for funding the response and cleanup operations, but vowed to increase federal involvement. [includes rush transcript]
Paramilitaries Kill Two Human Rights Activists in Oaxaca
In Mexico, two human rights activists have been shot dead in the state of Oaxaca. The victims have been identified as Beatriz Cariño, director of the Mexican human rights group CACTUS, and Jyri Antero Jaakkola, a human rights observer from Finland. They were traveling as part of a convoy attempting to deliver aid to a town that's been targeted by paramilitary blockades since the 2006 uprising against Governor Ulises Ruiz. [includes rush transcript]
Dorothy Height (1912-2010): Civil Rights Leader Remembered for Lifelong Activism
President Obama, First Lady Michelle Obama, along with hundreds of mourners, packed into the National Cathedral in Washington, DC on Thursday to attend the funeral of civil rights and women's rights leader Dorothy Height. She died last week at the age of ninety-eight. We speak with Stanford University professor Clayborne Carson. [includes rush transcript]
UC Berkeley Student Senate Fails to Override Veto of Israel Divestment Measure
The student senate at the University of California, Berkeley, has failed to override a veto of a bill calling on campus officials to divest from companies that supply weapons that Israel uses in its occupation of the Palestinian territories. [includes rush transcript]
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CDC Unveils New Health Worker Protocols for Ebola
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Australian Police Drop Probe into 1975 Murder of 5 Journalists by Indonesian Forces
Oscar Pistorius Sentenced to 5 Years for Girlfriend's Killing, Likely to Serve 10 Months
Reports: Ukraine Used Cluster Munitions in Donetsk in Possible War Crime
Philippines: After Murder of Transgender Woman, Protests Target U.S. Forces Agreement
U.N. Warns of "Rampant" Sexual Violence in South Sudan
Man Suspected of Killing 7 Women in Indiana
WTO Rules Against U.S. Meat Labeling Requirements
U.N. Officials Condemn Water Shutoffs in Detroit
Report: U.S. Spent Millions on Social Security Benefits for Nazi War Crime Suspects
Alabama House Speaker Mike Hubbard Indicted for Corruption
NOAA: 2014 Poised to Be Hottest Year on Record
Climate Change Whistleblower Rick Piltz Dies After Cancer Battle
By Amy Goodman and Denis Moynihan
The headlines shift hourly between Ebola and ISIS. The question is often asked, "Should we put boots on the ground?" The answer is yes — but not in the Middle East.
Show for Oct 22, 2014
Video Appears to Show U.S. Arms Airdrop in ISIS Hands
U.N. Warns ISIS Assault on Yazidis May Be "Attempted Genocide"
WHO Plans Large-Scale Ebola Vaccine Trials for January
U.S. Imposes Ebola Restrictions on Travelers from West Africa
U.S. Cameraperson, Spanish Nurse Declared Ebola Free
Afghanistan: Opium Poppy Crop Hits Record High, Despite $7 Billion in U.S. Funds
North Korea Releases U.S. Prisoner
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An Unprofitable Disease: In the Political Economy of Ebola, Who Lives and Who Dies?Oct 16, 2014
Mexico's Missing Students: Were 43 Attacked by Cartel-Linked Police Targeted for Their Activism?Oct 15, 2014


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Feminism Is Not a Monolith and Neither is Double X
Our first week at Double X is drawing to a close. And we've heard all sorts of responses. We're not feminist enough. We're too feminist. We say we're not feminist but then we talk a lot about feminism . We (and Slate ) are ghettoizing women . First, I want to second my co-editors Hanna and Emily in what they wrote yesterday and today about why we wanted to create Double X and its relationship to Slate . Second, I want to take this moment to clarify some things about the disparate points of view you'll find on the site.
The spirit animating the site is the spirit of debate. We do not edit the blog posts before they go up, or read them over to make sure they all hew to a single party line. And so XXFactor blogger Susannah Breslin, for example, may have one take about feminism , while our essays reflect another. When we at Double X have said that we'll have a "feminist" viewpoint, we do not mean that this viewpoint will be doctrinaire or singular-or even that every piece will have a "feminist" angle. For example, for our launch, we asked a range of women to answer our question, "What is the primary problem facing women today? What is today's problem that has no name?" Many of the essayists in our symposium chose to point to the problems with feminism itself. We did not coach the responses, or set out to hack feminism off at the knees. The essays reflected the writer's own views.
And that's, in our view, as it should be. We created Double X so that readers and writers would get to hear women's voices raised in cacophonous debate-not in well-oiled agreement. As editors, we believe in the importance of discussing issues of women's equality and identity, and we are not afraid of the word itself. But some of our bloggers and contributors might not agree with us. Maybe they'll be cacophonous and contradictory for a while. So be it. That's the reality of where we are the moment. Let the arguments continue.
Forget Oculus Rift
This $25 cardboard box turns your phone into an incredibly fun virtual reality experience.
The Congressional Republican Digging Through Scientists' Grant Proposals
Renée Zellweger's New Face Is Too Real
Sleater-Kinney Was Once America's Best Rock Band
Can it be again?
Whole Foods Is Desperate for Customers to Feel Warm and Fuzzy Again
I'm 25. I Have $250.03.
My doctors want me to freeze my eggs.
Smash and Grab
Will competitive Senate contests in Kansas and South Dakota lead to more late-breaking races in future elections?


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Tory tax 'cuts' may not offer much escape from higher-rate tax demands
Cameron's electioneering tax giveaway announced ...
My patience is wearing thin with the rogue lenders who still won't play fair
Payday lenders have been warned to get their act...
Did the UK economy do well after all?
Not when set in a domestic historical context. A...
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By Gillian Orr | Digital Digest | Thursday, 17 March 2011 at 11:07 am
The battle to keep the revolution alive
During the Egyptian revolution, some of the most inspiring scenes to emerge from Tahrir Square were the scenes of solidarity between Muslims and the Coptic Christian minority. Egypt's Copts were particularly discriminated against under Mubarak rule, so many hearts were warmed by images of Christians forming human barriers around Muslims as they lined up for [...]
By Jody McIntyre | Notebook | Thursday, 17 March 2011 at 9:49 am
Online House Hunter: Through the Looking Glass
Our Online House Hunter makes a move with Alice in Wonderland to find a home with links to the author Lewis Carroll
By Alan Cleaver | Property | Thursday, 17 March 2011 at 7:00 am
iBet: Dynamo Kiev will park the bus at Manchester City
Manchester City V Dynamo Kiev, Europa League, tonight 6pm
Lay over 3.5 goals at 5/2 with Betfair
Man City need to attack after losing the first leg 2-0 against the Ukranians. They will probably win, but they have not looked like a side that can easily put teams who defend deeply away in recent [...]
By Gareth Purnell | Sport | Thursday, 17 March 2011 at 1:00 am
"Son of Roadblock"
I thought Ed Miliband did quite well at Prime Minister's Questions because David Cameron seemed not to be answering his point about European competition law applying to the NHS. The Prime Minister seemed to think that it was enough (a) to point out that Labour's manifesto, written by Miliband, supported competition in the NHS; and [...]
By John Rentoul | Eagle Eye | Wednesday, 16 March 2011 at 4:47 pm
In it for the long run: head to the hills
Last Wednesday I was dodging green traffic lights and wobbly paving stones at the start of the A1 on a typical urban run. But by Friday afternoon I was running up a hill in three inches of snow, enjoying crystal clear skies with only the odd deer or cairn to distract me.
This was because I'd [...]
By Jane Bainbridge | Sport | Wednesday, 16 March 2011 at 4:28 pm
England's World Cup struggles: Pitches or players?
We look at the England team and think what lies around the corner for them. Will they produce heroics and rebuild their confidence after morale-crushing defeats to Ireland and Bangladesh? Or will the fans be left with another bitter taste in their mouths as the tournament reaches a premature end?
By Alexander Penny | Sport | Wednesday, 16 March 2011 at 2:12 pm
Caught & Social: Happy birthday to i
Daily gossip from the world of celeb
By Luke Blackall | Notebook | Wednesday, 16 March 2011 at 2:02 pm
Age Of The Dragons – DVD Trailer
Age Of The Dragons starring Danny Glover and Vinnie Jones is an all-action re-telling of the classic story like never before. Captain Ahab (Glover) and his crew hunt the most feared creatures to have ever existed: dragons.
By Inside film | Film | Wednesday, 16 March 2011 at 1:37 pm
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Pendleton Woolen Mills has tapped a former Filson executive as its new CEO.
Mark Korros will take over as CEO of the family owned Portland-based apparel retailer from Bill Lawrence, who will stay on as an adviser during the transition.
Korros joins from the Seattle-based outdoor outfitter Filson, where he was president and CEO. Korros previously held executive positions at Samsonite, Franklin Covey and Reebok.
Mort Bishop III, who represents the fifth generation of family management at Pendleton, will remain president.
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Silvandersson Sweden AB has selected Bug Off Pest Control Center to distribute its Cryonite System.
NEW YORK - Silvandersson Sweden AB has selected Bug Off Pest Control Center to distribute its Cryonite System.
Introduced in 2008, Cryonite makes use of carbon dioxide to freeze insects to death. It is a unique, non-toxic and easy-to-use technology that eliminates all life stages of crawling and flying insects like roaches, bed bugs, stored product pests and wasps, according to the manufacturer. Cryonite can be used almost anywhere: food establishments, hotels, hospitals and schools. It can be used directly on furniture, bedding and even electronic equipment, the manufacturer reports.
Silvandersson Sweden AB will be demonstrating the Cryonite System at the New York Pest Expo on Nov. 8, 2012, in New York City. Both wand options: The Rifle and The Lance (the extendable version) will be available for purchase at a deep discounted price at the Show.
For more information on Cryonite or to register for the New York Pest Expo, please call 212/781-2304 or visit www.bugoffpccenter.com.
The Scotts Miracle-Gro Company announced that its subsidiary EG Systems, Inc., doing business as Scotts LawnService, has signed a definitive agreement to acquire the assets of Action Pest Control, Evansville Ind., which ranked 56th on the 2014 PCT Top 100 list, with annual revenues of $11.6 million. The transaction, which is expected to close by January 2015, would mark Scotts' first acquisition of a structural pest control business.
Trade group Wildlife Control Business Builders (WCBB) is raising funds to help ECO Wildlife Solutions' Susan Sims, who suffered serious injuries as a result of a ladder fall.
Columbia University researchers spent a year collecting 133 rats in Manhattan. They found that the specimens were carrying food-borne illnesses like salmonella, as well as fever-inducing illnesses.
More than 3,000 industry professionals from 80-plus countries have traveled to Orlando, Fla., for PestWorld '14. The Opening Ceremony, sponsored by Bayer, featured musical act Rhythm Extreme.
Fortson, longtime president of Terminix Service, Columbia, S.C., was announced as recipient of the NPMA Pinnacle Award, NPMA's highest honor.


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Tim McGraw will release the single "Truck Yeah" to country radio programmers on July 3, marking his debut for Big Machine Records. McGraw is performing the upbeat song on the Brothers of the Sun tour with Kenny Chesney this summer. The song was written by Chris Lucas, Preston Brust, Chris Janson and Danny Myrick. "We have been unbelievably excited about 'Truck Yeah' since the first show of the summer when we played it live for the first time," said McGraw. "I'm really happy that radio and the fans will have access to it so soon."
- Florida Georgia Line, The Band Perry Earn Double-Platinum Singles
- Ariana Grande, Meghan Trainor Added to CMA Awards
- Jason Aldean's Old Boots, New Dirt Is Nation's Best-Selling Album
- Jason Aldean, Tim McGraw Face Awkward Questions
- Eric Church, Florida Georgia Line, Kacey Musgraves Added to CMA Awards


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Do you want to be part of a long term, consistent, and competitive Madden 15 PS3 league where most importantly you don't need to worry about member activity or quality? Join us and enjoy Madden 25
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Hey, It's Lynch Leave me a message (beep) Pick up, you there, Alright, Dude give me a call, I don't know what happened last night, Just let me know what's going on, I was wasted, Did we have a good time, give me a call, bye
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Written on: 31/10/2013
The staff was friendly to take my trouble ticket and giving me thier names and email addresses. But that is it their tech support could not fix their servers after 3 days of down time i had to switch to a different company. Because i walked away from them i could not get any my money back.
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Clone Trooper Training Academy, a favorite attraction of Marty Sklar
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Well, I met a lot of great people in here! There are definitely some knob heads, but without them, it would be boring! Got to love Teen Chat, it takes away all boredom.. It's the greatest time killer ever.
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This traditional hot and sour soup needs a soft and fruity white to match, which while not heavy in body, should have real intensity of flavour. We had the most success with Semillon/Sauvignon Blanc blends and Western Australia's Margaret River is home to this country's best examples of this classic style. It's worth noting that you do need to be careful of wines with excessive acidity – both here and with any of these dishes - as too much acid in a wine has a habit of making spicy food seem even spicier. Keep an eye out for producers such as Evens & Tate, Vasse Felix, Cape Mentelle and Cullen.
Bring together the scud chillies, sliced lemongrass, ginger, coriander root, kaffir lime leaf and the shallot in a mortar and pestle and bash to bruise and combine all the ingredients without taking it all the way to a paste.
Bring the stock to the boil and then reduce to a simmer, add the ingredients from the mortar and pestle and allow to simmer for about 5 minutes.
Taste the broth and then add limejuice, fish sauce and chilli paste until there is a balance between hot and sour. Once you have the balance to your liking add the prawns and straw mushrooms and simmer for a further minute or until the prawns are cooked.
Divide the prawns between the bowls and ladle the hot broth over the top.
Sprinkle the chopped coriander over the top of each soup and serve immediately.
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Riesling, Pinot Gris and Pinot Noir are the best wines to pair with Tom Yum Gung.
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Recipe by Tobie Puttock from Tobie & Matt: Asia
Recipe by Tobie Puttock from Tobie & Matt: Asia
Darwin ---the capital city of the Northern Territory Darwin has a pace that might - almost - be described as brisk, at least by Northern Territory standards. The city's populace now reflects its proximity to Asia: the mix of some 50 cultures including Aborigines, Vietnamese, Filipinos, Malays, New Guineans, Pacific Islanders, Japanese, Indonesians and European Australians, provides a strong cosmopolitan flavour. The Northern Territory's capital, Darwin was founded in 1869, after more than 40 years of failed settlements in the north – abandoned one after another because of malaria outbreaks, cyclones, Aboriginal attacks and supply failure due to the sheer distance from the other white settlements. It was named after Charles Darwin, one of whose shipmates on the Beagle discovered the bay in 1839. The best way to appreciate Darwin's multiethnic mix is to visit this market, held every Thursday and Sunday evening from April to October, with stalls selling foods from around the world and handmade craft including crocodile products, indigenous art and jewellery. If you have come to Darwin city, you will understand the local culture and history of Darwin, taste some delicious food and enjoy the amazing scenery here ,also take some photo is a pretty good experience . do you think so. If you can ,take the photo to the profession canvas prints shop to made a canvas painting, it is can as a gift to your friends or hangs in your home to add more beauty to your home. My Canvas Prints-Canvas Prints http://www.mycanvasprints.com.au/home/
» 17h ago


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Posted Apr 07, 2012By mleyba
OAKLAND, calif. — Though it came on the road and as the second game of a back to back, it would be tough to call the Nuggets' 112-97 loss to Golden State anything other than the obvious. Another opportunity squandered by a team fighting for its increasingly complicated playoff life. A win Saturday would have moved Denver (30-26) into the sixth spot in the Western Conference playoff race by itself with 10 games left to play. Instead, the Nuggets left Oakland with a tenuous hold on the eighth spot and staring up by half a game at Houston and Dallas.
Read more: Nuggets lose to Warriors, 112-97. Currently in eighth place in conference.
[insertSlideshowjava xml="http://director.denverpost.com/images.php?album=234" api="dpmc"]
The Denver Nuggets host the Oklahoma City Thunder in their NBA preseason opener at the Pepsi Center in Denver, Colo
The Denver Nuggets held training camp at the Pepsi Center in Denver, Colorado October 1, 2014.
Chauncey Billups announced his retirement from the NBA after 17 seasons of professional basketball.
Steve Ballmer is officially the owner of the Los Angeles Clippers. The NBA announced in a statement Tuesday morning
Former Colorado State star Becky Hammon was hired as an assistant coach by the San Antonio Spurs, the team announce
At a press conference, the Los Angeles Lakers introduce former player Byron Scott as their new head coach. Email st


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Much smaller than expected. Room enough only for very small toddler to sit. Quality is great - it's just small
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Our homemade sandbox needed some reinforcing at the corners and we were going to make seats from cut wood. When I saw these, I immediately purchased two and they are ideal. They reinforce the corners and they are better than wood seats since they aren't susceptible to insects and rot. Love the drain holes as well!
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perfect little sturdy seats for the sand box. Attachment was easy- no screws included, just bought some weather-resistant deck screws.
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Weather report: Cool across the central, northern Plains
In the West, frost and freeze warnings are in effect this morning in parts of the interior Northwest, including southeastern Washington and northeastern Oregon. Cool weather also covers the remainder of the region, except for warm, windy conditions in parts of the Southwest.
On the Plains, warm weather prevails in much of Texas, but cold air covers the remainder of the region. Snow lingers across eastern Montana and much of North Dakota.
In the Corn Belt, snow is falling across the far upper Midwest, including the Red River Valley. Elsewhere, isolated rain showers are occurring in the vicinity of a cold front stretching from Michigan to Missouri. Warmth in advance of the front's passage contrasts with a return to or continuation of cold weather farther west.
In the South, warm, dry weather in most areas favors fieldwork and crop development. A few showers linger, however, in the Atlantic Coast States.
Outlook: A storm system currently centered over Minnesota will move northeastward into eastern Canada. Snow will subside later today over the north-central U.S., but rain showers will linger through Tuesday from the Ohio Valley into the Northeast. Meanwhile, a stronger storm will begin to take shape across the nation's mid-section, resulting in substantial, late-season snow accumulations from the central Rockies into the upper Midwest. In addition, the latter storm will result in mid- to late-week downpours (locally 2 to 4 inches or more) from the southeastern Plains and Mid-South into the Midwest; severe thunderstorms from the central and southern Plains into the Southeast; and high winds from the Southwest to the southern Plains. In the storm's wake, temperatures could fall below 32°F as far south as the southern High Plains, and below 20°F on the central High Plains, on April 18-19. The NWS 6- to 10-day outlook for April 20-24 calls for below-normal temperatures in most areas from the Plains to the East Coast, while warmer-than-normal weather will prevail in the West. Meanwhile, drier-thannormal conditions across the majority of the U.S. will contrast with near-normal precipitation along the Canadian border and above-normal precipitation in the Atlantic Coast States.
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Murder investigator in the media
Prominent murder investigator Detective Senior Sergeant Ron Iddles is in the spotlight after reports by Fairfax Media today that he has been asked to move on from the homicide squadJanuary 14, 2014 6:39pm
The Bendigo Advertiser and The Age reported today that Sen Sgt. Iddles has been moved from the homicide squad less than a month after his controversial decision to use a graphic photo of Jill Meagher's body at a fundraising event.
The Riv understands the photo was used at a fundraising event in Echuca at one stage.
The reported plan to move him on is said to have infuriated colleagues of the 30-year veteran.
But in a statement provided to the Riv today from Victoria Police, it says there has never been any intention to move Sen Sgt. Iddles out of the homicide squad.
"It's not uncommon for managers and staff to meet and have discussions from time-to-time. The content of those discussions is entirely confidential," a Victoria Police spokesperson said.
"We don't intend to provide any further comment."
The Riv understands Sen Sgt Iddles grew up in Rochester and has been the face of a number of high-profile murder cases.
Thankyou for your comment.Your comment will appear once it has been approved by MMG staff.
Teens arrested after pursuits through Shepparton
Two teenage boys have been arrested following a series of traffic incidents in Shepparton last night.
Huge weekend set for Yarrawonga Mulwala Multi Sport Festival
Fine weather and a strong number of athlete registrations will ensure this weekend's Yarrawonga Mulwala Multisport Festival is another huge success.
Open garden party
The fifth annual King Billy Retreat Open Garden Party will be held at Rushworth on Saturday and Sunday, October 25-26, from 10 am to 4 pm.
New chair based in Echuca
The head of the Building Designers Association of Victoria has big plans for the association.
Kidzcapers fun in Rochester
St Joseph's Primary School students have been preparing their push carts ahead of next month's KidZcapers.
Actively training their workers
KCLC supported Southern Processing win award
Seymour and District Historical Society turns 50
Members celebrate over the weekend.
Flood funds for Berrigan Shire
Berrigan Shire was this week approved for a low interest loan from the state government to undertake flood mitigation works.
Grey Fergie returns home
Frank Hill is thrilled with his repaired 1948 grey Fergie.
Cobram Police: remember to lock up
As weather warms, so does car theft; Cobram police urge drivers to lock parked vehicles.
Funds raised for McHappy Day
Deniliquin McDonald's raised more than $1300 for McHappy Day on Saturday.
Fruit research day at Tatura
The latest in stone and pome fruit research and development will be the focus of a forum at the Horticulture Centre of Excellence at Tatura tomorrow, Wednesday, October 22.
Benalla vision impaired woman finds new independence
Lions Benalla donates $1200 to buy a bar code reader for Lorraine Billman who will soon be able to go shopping without her husband.
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Factice Perfume Bottle
Victorian Domed Butter Dish
Angel Skin Coral Cameo Stick Pin
Chinese Enamel Silent Butler
Lead Crystal Lustres
Artist Signed Limoges Plate
Austrian Portrait Cream Jug
Hand Painted Nippon Bowl
The Most Beautiful One
Lounging As Usual
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WESTCHESTER COUNTY, N.Y. -- A man charged with driving in the wrong direction and nearly crashing head-on into a police car was intoxicated, according to a report on LoHud.com.
The incident occurred on the southbound Saw Mill River Parkway at around 3 a.m., report, and the driver, 40-year-old William Bratton of Nyack, failed a sobriety test and faces a variety of other charges, according to the report.
- 1 Michael Douglas, Catherine Zeta-Jones Purchase New Bedford Home
- 2 Bedford's First Snowfall Of Season Could Arrive Over Weekend
- 3 Superstorm Sandy Was Not The 'Big One,' So Bedford Must Prepare
- 4 Police Issue Alert For Missing Teens Who Could Be In Westchester
- 5 Police Search For Suspect Who Allegedly Robbed Man Near Bedford


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Very Good Recipes of Spinach - 13
There are 1262 Spinach recipes on Very Good Recipes.
Page 13 out of 50.
Do you like spinach recipes?
Click on the title of a recipe or the photo of a dish to read the full recipe on its author's blog.
Recipes by date | Most popular recipes
Aloo Palak Recipe | Aloo palak gravy recipe | potato spinach curry By SpiceuptheCurry 02/25/14 01:44 Preparation: 15 min - Cooking: 30 min Aloo Palak curry - very delicious and mild flavored Indian curry recipe made with potatoes and spinach with very few spices. There are two ways of making aloo palak – one is dry aloo palak subzi and other is aloo in palak gravy. Today's recipe is gravy... Add to my recipe book
Spinach Salad w/ Honey-Poppy Seed Dressing By The Café Sucré Farine 02/24/14 17:02 I love finding treasures, how about you? To me, they often come in the most unexpected places ....... like this delightful set of salad bowls. You'll never believe where I found them ........... READ THE REST OF THIS ENTRY> Add to my recipe book
Spinach Salad w/ Honey-Poppy Seed Dressing By The Café Sucré Farine 02/24/14 17:02 I love finding treasures, how about you? To me, they often come in the most unexpected places ……. like this delightful set of salad bowls. You'll never believe where I found them ……….………. Scott and I did a bit of exploring last week, discovering an area... Add to my recipe book
141.6…Hot Spinach and Artichoke Dip By Melissa Likes To Eat 02/23/14 23:02 It was another successful "meeting" of our Gourmet Club last night! I was in charge of bringing the appetizer. I went with something simple and delicious: Hot Spinach Artichoke Dip. I figured I couldn't go wrong with this. I was right. It turned out delicious!... Add to my recipe book
Potato Spinach Enchiladas By Vegetarian Underground 02/23/14 22:38 Potato and Spinach Enchiladas Potato Spinach Enchiladas I made these enchiladas for a family gathering yesterday, and they were enjoyed by all. They can be assembled a few hours ahead, refrigerated, and cooked when guests arrive. Serves 8. Ingredients:Sauce:12... Add to my recipe book
Steamed Cod With Spinach By Wholesome Ireland 02/23/14 21:20 Every Friday, we get fish in Howth. If the fishing has been good then we'll get enough for a few meals over the weekend. If not, then it's simply a white fish for one meal. 52 weeks of the year we eat fish at least once a week. We are the third generation... Add to my recipe book
White bean, spinach, and pecan timbales By Out of the Ordinary 02/21/14 19:18 I've just discovered that a "hope" is a piece of enclosed land in the midst of fens or marshes or of waste-land generally. (OED) It's also a small, enclosed valley, or an inlet, a small bay or a haven, you'll … Continue reading → Add to my recipe book
Spinach, ricotta, saffron and fennel cakes By Deena Kakaya Vegetarian Cooking and Reci... 02/21/14 09:33 This week I've been on my own with the boy, again. His daddy was in the states for work and so our week went with the usual mayhem of arts and crafts tumbling onto forbidden zones, the boy insisting on only pooping in the bath, his cupping my face and saying,... Add to my recipe book
Chana dal khichdi with spinach and tomato /चना दाल खिचड़ी टमाटर और पालक के साथ By Hema's Musings 02/20/14 10:53 खिचड़ी उरद दाल की हो या फिर अरहर ,चने की कभी- कभी खाने में अच्छी लगती है ,और अगर साग के साथ बनायीं जाये तो और भी स्वादिष्ट लगती है। हम अक्सर वीरवार को चना दाल ही खाया करते हैं वो फिर चाहे दाल बनाकर खाएं या चना दाल की खिचड़ी ---आप भी ट्राई करिये --- सामिग्री---चावल... Add to my recipe book
Palakura Pesarapappu – Spinach Moong Dal Curry By Swasthi's Indian Food Blog 02/20/14 09:25 When it comes to making a dal for meal, I always prefer to make a smooth and creamy dal like dal pumpkin (gummadikaya pappu), sindhi kadhi with ladies finger or dal tomato (pappu tomato), where all the ingredients can be dumped to cook in the pressure cooker... Add to my recipe book
Palak Paratha |Spinach Paratha By Desi Fiesta Desi fiesta 02/20/14 06:36 Parathas are always an yummy way to fill your tummy . Instead od stuffed parathas now a days we love to have plain flavored paratha's . Whole wheat flour is a very healthy way to start a day and so paratha's are served for breakfast too. I made spinach... Add to my recipe book
Spinach Pepperoni Bread By 365 Days of Baking 02/20/14 05:14 Spinach Pepperoni Bread - A rolled bread filled with chopped spinach, pepperoni slices and provolone cheese. It makes a great dinner or the perfect finger food for Game Day! Day 19. Can you believe it? I can't. I've been baking for almost three weeks now.... Add to my recipe book
Skinny Spinach Tart with a Squash Crust By Hungry Couple 02/19/14 14:08 Quiches and tarts and hand pies...sweet or savory....I love pretty much all foods in a crust. The trouble is that, no matter how much you skinny up the filling, there's all that butter in the crust. I have made olive oil doughs before that are tasty and... Add to my recipe book
Spinach stir fry By Cook click n devour!!! 02/17/14 18:26 Unlike most of people I know I am very fond of greens.Only thing I will always feel really lazy to clean and cut them as I feel it is a tedious process.Spinach is quite easy to clean and cook compared to few other greens and also we love the taste of spinach.When... Add to my recipe book
Chicken with Spinach Patties and Condiments form Victoria Amory & Co By Living the Gourmet 02/16/14 20:39 Living the Gourmet is pleased to present the all natural, handcrafted condiment line from Victoria Amory & Co. Living the Gourmet had the opportunity to sample three of the condiments, the Champagne Ketchup, the Classic Lemon Mayonnaise, and the Red Chili... Add to my recipe book
French lentil and butter bean soup with tarragon and spinach By Out of the Ordinary 02/16/14 16:46 Reverend Gary Davis was born in Laurens, South Carolina in 1896. He was born partially blind, and became fully blind as an infant. He was the only one of eight children to survive to adulthood. His father was killed when … Continue reading → Add to my recipe book
Spinach Spelt Salad with Cranberries and Carrots for #TheSaladBar By Shockingly Delicious 02/15/14 11:33 Chewy, complex, nutty spelt joins garlicky onions and carrots, pine nuts and pomegranate for a gorgeous winter spinach salad. Add to my recipe book
Nadan Cheera Thoran / Spinach Stir Fry By Cooking is easy 02/15/14 09:34 Nadan Cheera Thoran or spinach stir fry with coconut is an everyday side dish for rice. In Kerala, cheera (a variety of spinach) is grown in the backyards, most popular are the "red cheera" and the "green cheera". Now-a-days we get them in local vegetable... Add to my recipe book
Spinach and Portobello tarts with a pecan crust By Out of the Ordinary 02/14/14 21:41 We both forgot it was valentine's day. To be honest, I also forgot it was Friday or mid-February and I can barely remember my name. It's all a big blur of snow and sniffles and boys home from school every … Continue reading → Add to my recipe book
Palak Anda / Spinach Egg Curry By BlogExplore 02/12/14 07:37 Preparation: 25 min - Cooking: 10 min Palak anda / spinach egg curry recipe is an Indian style curry of boiled eggs in a spinach gravy. I had a bunch of palak leaves and was on the verge of making chole palak but settled on trying out this gravy which turned out delicious. I love the vibrant... Add to my recipe book


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Articles that are news items
There's Another Name on Top of the QMJHL Scoring RaceSubmitted by chad on November 12, 2005 - 10:42
As reported last night, Stan Lascek had four assists for Chicoutimi in a 6-0 win over PEI. With the four points, and only a goal and assist from teammate Maxime Boisclair, Lascek pulled into a tie with Boisclair for the QMJHL scoring lead with 49 points in 22 games (17-32-49). Lascek was the game's first star. Speaking of Boisclair, in a recent edition of The Hockey News, Boisclair states that Tampa Bay moved him from his natural wing position to center during the Traverse City Prospects Tournament in September where he was trying out for the club, and because of the unfamiliarity with the position, Boisclair was extremely unhappy with his impact in the tournament. Boisclair was not offered a contract by the Lightning, but hopes to attend another NHL camp in the fall.
- Read more
- 636 reads
Lundin, Smaby Notch Assists in NCAA Action FridaySubmitted by chad on November 12, 2005 - 10:34
With the exception of Arthur Femenella, all Lightning NCAA prospects were in action Friday night. Femenella's team, the University of Vermont, did indeed play, but Femenella was scratched. Highlighting the night for Baby Bolts was Mike Lundin of Maine, who had an assist in a 9-2 Hockey East win over UMass-Lowell, and Matt Smaby, who had an assist as North Dakota beat WCHL opponent Minnesota Duluth 5-3. Also, P.J. Atherton, though scoreless, had a plus-two rating for Minnesota, who crushed Alaska-Anchorage 9-0. John Wessbecker (UMass), and Dusty Collins (Northern Michigan) both went scoreless i
- Read more
- 544 reads
Game Night 11/11: Titans at ChiefsSubmitted by pete on November 11, 2005 - 22:48
And the Chiefs still haven't won a game. TRN-3 JHN-2 (SO) It's good to see Justin Kelly in the lineup and scoring a goal for the Chiefs. He's a player Johnstown definitely needs. It's also good to see Chateau du Bow Wow's penthouse suite occupant, Adam Henrich, get a goal tonight. Still, Morgan Cey's pro debut was ruined in the shootout despite stopping a staggering 26 of 28 shots faced. The good news is with Kelly and Henrich in the lineup along with Greco and Egener already in place, the Chiefs managed 44 shots on goal tonight, perhaps showing signs of turning the corner. Game sheet from [url=http://www.echl.com/cgi-bin/ppublic.cgi?action=summary&id=2380]ECHL.com.[/url]
- 591 reads
Game Night 11/11: Falcons at PiratesSubmitted by pete on November 11, 2005 - 22:45
Ahhhhh, it's like a deep dark soulless pit of despair right now... SPR-1 POR-5 Brian Eklund gets torched for five goals as the Falcons, who were no doubt emotionally shellshocked after last night's collapse against Manchester, got roughly doubled up on shots. Game sheet from [url=http://stats.theahl.com/stats/game-summary.php?game_id=1001199]TheAHL.com.[/url]
- 660 reads
Game Night 11/11: Lightning at ThrashersSubmitted by pete on November 11, 2005 - 22:28
OK, OK, is this finally rock bottom? TB-2 ATL-5 I would've preferred Gerald Coleman's debut had come under better circumstances. The Tampa Bay Lightning just blew a two goal lead and allowed themselves to be beaten by an ECHL level goaltender. That's the plain, unvarnished truth. I have no doubt that the word of closed door players only meetings is right around the corner. There needs to be a "come to Jesus" for this team. And it's clear to me that it is no longer Dave Andreychuk and Tim Taylor's ship to fix. They have fulfilled their charge to nurse a bunch of kids into an NHL contender and Stanley Cup champion. Those kids are all grown up, and those kids are the ones who now need to step up and take ownership of what is happening to this team. There are about $15 million dollars being tied up in three players on this team: Lecavalier, St. Louis and Richards. We can talk about the unstable goaltending or the fact Darryl Sydor couldn't defend his way out of a wet paper sack right now, but that ignores the obvious. In all of these games where the Lightning knew going in they had a goaltender with flagging confidence or a wet behind the ears rookie between the pipes they still only managed to score a couple of goals against Atlanta, New York and Montreal. The timely goal scoring has simply not been there. Much of the reason for that is that the Tampa Bay Lightning simply have not made the commitment in moment-of-truth situations in this six game losing streak to make the hard, dirty play. To shoot the puck and crash the net. This team is still looking for answers in tic-tac-toe plays that never work when you're struggling instead of putting on their hard hat, grabbing their lunch pail and doing what it takes to get points in crunch time. It's time to start making that commitment again. It's time to start talking about accountability again. But the one's stepping up in those closed door meetings to talk about accountability need to be the $15M men. It's time to grow up. It's time the torch was passed. It's time that Vincent Lecavalier no longer leads the team in zeros and the end of his check but also he leads vocally in the dark times. And no, I'm not blaming Vinny or saying he's dogging it. Quite the contrary, he's one of the few players on this team playing decent hockey right now. But he can't just sit back anymore while Dave Andreychuk and Tim Taylor do all the heavy lifting in the locker room. Nor can Brad Richards. Nor can Martin St. Louis. This is their time, and if things are going to turn around they need to make this, unequivocally, their team. Right now. By the grace of God, this team plays in such an awful division 2 games under .500 can still find you in second place ahead of Florida. But that won't last forever. It's time to get back to work.
- 679 reads


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|Photos of Brides
In this wedding portfolio gallery I have selected a couple of my favourite photos of brides. I focus most of my attention during the wedding day on capturing the bride in the most beautiful way I can. This is what makes my husband's assistance so crucial - while I focus on the bride, he will capture the groom. When the bride and groom is together, we work our magic together to make sure the wedding couple shines in front of our cameras. If you want to book me for your wedding you can download my wedding photography packages and contact me to find out if I'm available.
Photography Tips
Beyond The Wedding Album
You hired a wedding photojournalist, but when it comes to deciding on the best way to share your story, the choices might overwhelm.
Wedding Tips


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Submitted by Dean C.
Your Honor, the prosecution moves for the death penalty and rests.
That just about wraps it up for another edition of AwfulVision! Thanks to everyone who submitted some more stupid bullshit for me to watch! Your Official AwfulVision Bags of Dicks (for you to eat) is in the mail. If you'd like to join in on the dick-eating fun, feel free to email a terrible video to me (or post it on our wall) and delight in the fact that you've made my life just a little bit more unbearable. See you jerks next time!
Call of Duty Advanced Warfare promises to up the ante on Kevin Spacey's face in a video game.
Take a journey to the haunted end of the uncanny valley with these terrifying and just plain terrible youtube videos for children.
After 34 episodes the Gaming Guyz FINALLY hit their stride!... in the final episode.
The SA Goons create a new batch of adorable/abominable animal hybrids for the first time since 2008!
It's too late to buy Boss's Day gifts, you're fired. But you can still shop for other fake holidays!
As you may have noticed, the most popular viral videos at any given time are amazingly banal, annoying, and cliched pieces of waste. It almost seems as if the internet naturally gravitates towards the worst possible Youtube and Google video selections. So it stands to reason that if the terrible videos become popular, then the unpopular videos must be awesome! We here at Something Awful present to you AwfulVision™, our own patented service dedicated to showcasing a wide selection of unpopular videos that apparently must be good! Welcome to Web 3.9. Welcome to AwfulVision™!
Copyright ©2014 Rich "Lowtax" Kyanka & Something Awful LLC.