Sunday, January 31, 2010

New Bugatti Veyron Modification 2010

New Bugatti Veyron ModificationThe All New 2010 Bugatti Veyron to Feature the W16 Engine and 4 Turbochargers!
The Veyron from the house of Bugatti is expected to come in the 16 cylinder W16 engine mounted in separate two banks of eight cylinders. The engine features with the 4 turbochargers and displaces 8.0 liter with the stroke and the bore of 86 mm and the 86 mm respectively. The vehicle is designed by Ricardo.
new bugatti veyron red blackThe transmission in the vehicle consists of Direct Shift gearbox with dual clutches, manual gearbox controlled by the computer with the 7 gear ratios, magnesium paddles next to the steering wheel and has the shift time of 150 Ms. The vehicle uses the Haldex traction system and provides the 4-wheel drive. The new Veyron uses the flat tires from the Michelin, which are specially designed for the Veyron to provide the maximum speed. The curb weight of the vehicle will be 2034 kilo grams.
Bugatti Veyron modificationThe wheel base of the Veyron is about 2710 mm, while the length, width and the height of the model are 4462 mm, 1998 mm and 1204 mm respectively. The Bugatti Veyron modification has 10 radiators in total - 1 hydraulic oil radiator for the spoiler, 3 radiators for the engine cooling system, 1 engine oil radiator, 1 heat exchanger for the air-to-liquid intercoolers, 1 differential oil radiator, 2 for the air conditioning system and 1 transmission oil radiator.
Bugatti Veyron machineThe engine in the vehicle produces 746 kilo watts of power and a massive 920 lb ft of torque. The maximum speed of the vehicle is about 400 kilo meters per hour, and the most fascinating part of the story is that the new Bugatti Veyron is capable of reaching the speed of 200 kilo meters per hour in just 7.3 seconds.
bugatti veyron handling accelerator

New Red Nissan GTR Supercar 2010

nissan gtr red
Supercars have always existed to keep our passion for automobiles on constant boil, and supercars that don't crest the six-figure mark are the best kind. They're almost attainable. The RED Nissan GTR 2010 remains that way for 2010 with a price bump to help pay for a few upgrades from the factory. The base model will begin at $80,790 and the Premium model at $83,040. According to Edmunds.com, the new MSRPs are both $3,950 more than what you would pay for a 2009 model.

What does an extra four grand get you? A lot actually, not the least of which is a 5-horsepower bump for the twin-turbo 3.8-liter V6, which nets a new grand total of 485 hp. Other additions include a retuned suspension with redesigned Bilstein shocks, an upgraded braking system with more rigid brake lines, standard front seat- and roof-mounted side curtain airbags for the base GT-R model, standard wheels for the GT-R with a darker finish and "near-black" metallic wheels for the Premium model. A new color is also available, Pearl White, and you get a polished front bumper now when ordering your car in Super Silver.
Red Nissan GTR 2010Oh, and there's one more thing: Nissan has given the GT-R new Transmission Control Module programming. The new code is said to increase durability and drop acceleration times with the Vehicle Dynamic Control activated. It should also put an end to concerns about damaging the transmission by using the vehicle's Launch Control with VDC turned off. Preliminary reports indicate that the 2010 GT-R is still faster than all get out with the new programming.
PRESS RELEASE
NISSAN ANNOUNCES PRICING FOR 2010 GT-R
2010 GT-R RECEIVES HP BOOST TO 485HP

FRANKLIN, Tenn. (March 16, 2009) – March may be the month known for madness, lions and lambs, but at Nissan North America, Inc. (NNA), the biggest roar is coming from the early introduction of the new 2010 Nissan GT-R supercar, including a number of important enhancements. Specifically, five additional horsepower, a revised suspension, updated wheel finishes and standard front seat- and roof-mounted curtain side-impact supplemental air bags.

First introduced in the United States in July 2008, the red Nissan GT-R earned near universal acclaim, including being named Motor Trend "2009 Car of the Year," Automobile magazine's 2009 "Automobile of the Year", and winning Kelly Blue Book's "2009 Best Resale Value Award."
Nissan GTR 2010 RedFor 2010, the horsepower rating of the GT-R's 3.8-liter twin-turbo V6 engine has increased to 485 hp (from 480 hp) and the car's high-performance, 6-speed, dual-clutch transmission receives new Transmission Control Module (TCM) programming designed to optimize clutch engagement for improved drivability, and improve vehicle acceleration with the Vehicle Dynamic Control (VDC) on (activated). In addition, the braking system has been updated with more rigid brake lines for improved durability, and the brake calipers now carry both the Brembo and Nissan logos. Finally, the GT-R's state-of-the-art suspension has been retuned with redesigned Bilsteinâ shocks with a new valve body design and revised spring and damper rates.

The base GT-R model is now equipped with slightly darker, high-luster, smoke finish for the 20-inch RAYS forged aluminum-alloy wheels, while a new "near-black" metallic wheel finish is standard on the Premium model. For 2010, one new color – Pearl White – is offered, while the Super Silver exterior color has been enhanced to include a polished front bumper.

For 2010, the Nissan GT-R will again be offered in two models – GT-R and GT-R Premium. All 2010 Nissan GT-Rs are equipped with a standard 3.8-liter twin turbo V6 backed by an advanced paddle-shifted, dual clutch rear transmission and a world's first independent rear transaxle ATTESA E-TS all-wheel drive system. The Manufacturer's Suggested Retail Price (MSRP)* is $80,790 for the GT-R, and $83,040 for the GT-R Premium model. Destination & Handling (D&H) is $1,000.

Two options and two accessories are available for the 2010 GT-R: The Cold Weather Package (no charge); Special Super Silver Paint ($3,000); iPodâ Converter ($400); and carpeted GT-R floor mats ($280). Complete pricing information is available on the attached sheet.

The 2010 GT-R will be available only through officially certified Nissan retailers that have met a number of strict sales, service and facility commitments, including dedicating a master technician to GT-R service, on March 21, 2009. A complete listing of the nearly 700 GT-R Certified Nissan dealers is available to consumers on NissanUSA.com.

In North America, Nissan's operations include automotive styling, engineering, consumer and corporate financing, sales and marketing, distribution and manufacturing. Nissan is dedicated to improving the environment under the Nissan Green Program 2010, whose key priorities are reducing CO2 emissions, cutting other emissions and increasing recycling. More information on Nissan in North America and the complete line of Nissan and Infiniti vehicles can be found online at www.NissanUSA.com and www.InfinitiUSA.com. or visit http://modif-and-otomotif.blogspot.com/

Nissan GTR 2010 Modification

Nissan GTR 2010 modificationBeing an automotive journalist is like being a male porn star. We're little more than Piloti-shoed buffers between the reader and the objects of their lust, and really, no one cares about us. Still, you only get one chance to make an initial impression, so my first review here on Autoblog had to be big. As luck/fate would have it, I got a phone call a few weeks back that went a little something like this: "How'd you like to drive the first 2010 Nissan GTR on the West Coast, before the buff books get it?" Needless to say, the answer was obvious. But what to do with the brand-new R35, one of the most heavily and relentlessly covered car-stories of the past year? This takes us right back to that porno metaphor: How do I give the people what they want?
Nissan GTR 2010 Modif WallpaperWe hatched a plan – take the uber-Nissan down to San Diego and pay a visit to Comic Con! A story about 400-pound guys in Batman suits drooling all over the new GT-R practically writes itself, so we contacted various video game companies to see if they would let us drive the GT-R right onto the convention center floor. Perfect! Our stunt would be like lowering a nude, greased-up Megan Fox into a frat house. What could possibly go wrong? Without getting into the epic fail of that last bit, it didn't happen. What you're left with then is yet another review of a Nissan GTR 2010 Modification where some "pounding at 11/10s" wannabe hamfists Godzilla through envy-inducing, tight, twisty Southern California canyons. Lucky you... err, me.
modification of Nissan GTR 2010My task then would be to answer the following: There's endless talk about whether or not the Nissan GT-R 2010 modif has a soul. Yes, we all know it's supercar quick and hypercar capable. And yes, Japan's most recent foray into the segment can utterly dominate and humiliate most British, Italian and German machines – all costing two, three or five times as much – and give like-minded American all-stars a run for their ACR/ZR1 money. But is the GT-R anything more than a numb supercomputer, mindlessly parsing bits of data and then spitting out traction and velocity? Are its capabilities a credit to Nissan's mechanical engineers, or its electrical wonks? To put it another, more Comic-Conny way, is there a ghost in Nissan's machine?
Modification of Nissan GTR 2010The big news is bye-bye launch control.
First and foremost, we should cover what's new for 2010. The big news is bye-bye launch control. We found the GT-R's penchant for grenading transmissions humorous (from a distance), but alas, farewell. However... maybe it's still there? Maybe Nissan was only telling people launch control had been deleted? We found a very deserted stretch of road, put the transmission and suspension into R mode, turned the VDC all the way off, planted our left foot on the brake pedal and pushed the throttle with our right. Instead of the tach zinging up to 4,500 rpm, fuel cutoff happens right around 2,000 rpm. Launch control is deader than last Thanksgiving's turkey. That's not very soulful.

That said, the Transmission Control Module (TCM) has been reprogrammed. Not only can the six-speed dual-clutch gearbox shift faster (when in R), but the chances of a customer having to shell out $20,000 for a new cogswapper is greatly reduced. The 2010 GT-R also sports five more horsepower, bringing the total to 485, while torque output remains unchanged at 434 lb-ft. Rumors still persist that since each GT-R engine is hand-built, power levels vary and some engines churn out as much as 520 hp, if not more. Let's chalk this up to some engines running 100 octane and others dealing with California's crapola 91 high-test. Bottom line, the power feels freakishly adequate.
Nissan GTR 2010 SilverThe suspension's been retuned and the Bilsteins are a new design, while the brakes (somehow) have been revamped and fitted with more rigid lines and fresh pads. Our Premium GT-R tester arrived with dark, "near-black" wheels and when coated in Super Silver (like this car) you get a polished front bumper (there's also a new hue called Pearl White). More power, faster shifts, better handling, stouter brakes, blacker wheels and a transmission that's much less likely to eat itself? That sounds fantastic. Soulful, even.



You cannot fit your hand between the front and rear thrones.
As far as looks go, let's face it: You've been staring at the GT-R through your computer screen for as long as I have. Not a single body panel was changed for 2010, so you either love it, or you're indifferent. I will say this, Godzilla is huge. I knew the Nissan was a big boy, but it's nearly ten inches longer than a C6 Corvette; its wheelbase is fifteen inches longer than a Porsche 997. But hey, we were able to fit a case of wine and a large pizza in the trunk, so I'm sure owners aren't complaining too much about the GT-R's dimensions.
Nissan GTR 2010 modification pictureWell, maybe a little about the back seat. No joke: You cannot fit your hand between the front and rear thrones. Even children would be miserable. As far as the front of the cockpit goes, for a Nissan, it's pretty much okay. For an $86,000 vehicle, it's not nearly as nice as you'd like. Sure, there's leather all over the doors and on part of the dash, but it's budget, cheap-feeling leather, not that opulent, veal-fat rubbed and pleated cowhide you'd find in a Spyker. However, all that really matters is the usable stuff. The pedals, steering wheel, flappy-paddles, seats and handbrake are all up to supercar snuff. Especially that burly handbrake.



So then, how's it drive? In a word, Supercalifragilisticexpialidocious. From a standing start, the forward thrust is obscene. Pornographic, to stick with our earlier metaphor. 60 mph happens in less than four seconds (thanks to launch control delete, the GT-R no longer hunts in the 3.2/3.3-second Enzo/ZR1 woods) and the quarter-mile is annihilated in less than twelve. Trap speed? 120 mph, give or take. Top speed? North of 190 mph.

All these numbers are as quick or quicker than a $200,000+, all-wheel drive, paddle-shifted, 552-horsepower Lamborghini Gallardo LP560-4 or a $280,000 502-hp Ferrari F430 Scuderia. But forget about the numbers, the price tags and the competitor's badges. Instead, concentrate on the massive brutality taking place and hair-splitting wail of the turbos at full clip. You get to watch the scenery deform all around you as the buzzing builds to full cresendo. After giving one pal a quick blast up an empty freeway, he didn't want to shake hands because his palms were so sweaty. The GT-R is a face-puller, a neck-snapper, a pulse-pounder. Especially when you're banging off shifts in half a second with one of the world's finest paddle-shifted dual-clutch gearboxes. And that's in default. Pop the transmission into R-mode and the shifts are over and done with in two-tenths of a second. Bloody hell, indeed.

Nissan GTR 2010 ModifYeah, yeah, yeah -- 485 hp cars with 434 lb-ft of torque and AWD traction should be blisteringly fast when pointed straight. But the GT-R weighs over 3,800 pounds. Can the near two-ton porkster mechanically handle the twisty stuff? Simply put: Yes. But 'handles' is not even the right word. Dispatch, conquer, dominate, tear asunder, murder – that's it – the GT-R murders corners dead. Starting with the donk-sized tires (255/40 R20 in front, 285/35 R20 out back), moving to the retuned suspension and ending with the innards of a Swiss watch-fancy AWD system, the GT-R has world-class Stickum.

3,829-pound cars shouldn't change direction like Barry Sanders in his prime.
If I may quote McLaren F1 designer Gordon Murray after he climbed out of the Bugatti Veyron for the first time, "One really good thing, and I simply never expected this, is that it does change direction. It hardly feels its weight. Driving it on a circuit I expected a sack of cement, but you can really throw it at tight chicanes." Replace the word "circuit" with "stomach churning canyon road" and those are my sentiments exactly vis-à-vis the 2010 GT-R. 3,829-pound cars shouldn't change direction like Barry Sanders in his prime. Yet this one does.
Nissan GTR 2010 Interior ModificationDid I mention the stoppers? Going by the ancient caveat, "A car's only as good as its brakes," the new GT-R is the third best car in the world. Period. Cymbal-sized 15-inch discs at all four corners and six-piston Nissan-branded Brembo calipers join forces to mess with the space/time continuum. Slowing the car from 60 mph is near effortless. When you dip deeper into the near-bottomless well of power -- say around 140 mph – the brakes still work flabbergastingly well, hauling you down to something resembling a speed limit in mere moments. At one point in downtown L.A, a school bus cut us off and neither driver nor passenger were the least bit worried. Why? We'd been using the brakes all day – we knew.



Ah, but here it comes. Here's the part of the story where I'm supposed to tell you that yeah, you can hoon a GT-R harder and faster than a Lamborghini or a Ferrari. But the Italians are works of art, a living, breathing symphony filled with arias pointed right at the heart of an automobile where man and machine become one via harmony, divine intervention, etc. The GT-R? Nothing but a Silicon Valley automaton, precisely but passionlessly going about its servile duties while totally disconnected from the world around it and driver within. Well guess what? I'm not saying that – or anything resembling that. In fact, I'm going to say that those who complain about the GT-R's supposed soullessness simply aren't pushing it hard enough. Because once you do, you hear the angels sing. New rule: All observations concerning the GT-R at less than 80 mph or 5,000 rpm (whichever comes first) are meaningless. Under that and Godzilla's not even breathing hard.

When the gloves come off (transmission's in R-mode, suspension's in R-mode, VDC's in R-mode, nitrogen-filled tires are nice and warm and tacky) not only can the new GT-R rundown the aforementioned farm animals, but it's just as rewarding to drive – if not more so. Why? You can brake later, you can hold the road longer and you can blast out of corners quicker. Isn't that why we drive? Like many great cars, the GT-R seems to shrink when pushed, and the harder and meaner, the smaller it gets. Italian exhausts sound a thousand times better, but the GT-R is simply the better supercar. You'll be continually shocked that such a heavy, civilized and inexpensive car is not only capable of, but eager to rotate on its axis, accelerate like a rally car over busted pavement and keep its driver cool/comfortable after hours of use and abuse.



One caveat: during all of our testing we left the VDC on (except for our ill-fated attempt at launch control) and in R-mode for several reasons. The first being that Nissan strictly forbids operating the car with the VDC defeated unless you're stuck in snow or mud – it straight-up voids the warranty – and we didn't feel like returning a handful of broken half-shafts and shredded gears to the good folks at Nissan. The second reason being that most of our evaluative drives were on two-lane public roads with the high possibility of oncoming traffic and blind corners.

The GT-R inhabits the same plane of existence as Porsche's legendary 959.
That said, the VDC in R-mode has such incredibly high limits that we were able to register full back-to-back 1g lateral acceleration pulls in two different directions (as indicated by the g-meter oscilloscope) again and again and again. The car almost never lets go. And if it does step out (journo-speak for, "I entered the corner too hot, mashed the brakes while chopping the wheel and staring at a squirrel"), the sensation is akin to a hand reaching down from the clouds, grabbing hold and gently performing a quick course correction – the same way you used to play with your Hot Wheels. There's no sudden loss of power, no cruel nanny coming in like a guillotine. Just a little bit of wiggle, and you're back in the game, pushing the edge of the envelop, gunning for an M6, Ducati Monster, Dodge Viper and a few Ferrari F430 Spiders (yes, we did). Over the course of five days, five tanks of high-grade gasoline and more than 600 miles, we experienced the full force of the VDC saving our butt exactly once. And yeah, it was when we were trying to pull away from the damn Ducati.
Nissan GTR-2010So what's up then? Why the bum rap? Why do so many journalists (and I can't name names because I'm friends with so many of them) write off the GT-R as highly competent but ultimately soulless? Sure, it's one of the four or five most mindboggling performance vehicles on sale, but... meh. They just don't dig it. Besides not driving it hard enough, here's another explanation: Remember when compact discs came out? There was a seemingly endless series of interviews on MTV with guys like Tom Petty bemoaning the loss of the pops, hisses and scratches inherent to records. The flaws were part of the sound, man. You can't flip a CD over – how do you know what side of the record is playing? That's fine, but it's just knee-jerk reactionary nostalgia.

Let's look at the 2005 winner for supercar bang-for-the-buck: the Corvette Z06. It makes about identical power compared to the GT-R, but weighs 700 pounds less. Yet the GT-R is faster, both in a straight line and around corners. Stops better, too. Neat party trick, no? Yes, laying fat, smoky strips of incinerated rubber a hundred feet long is fun, but it's old fashioned, and in many ways, it's making the best of a compromised situation. On the other hand, the GT-R inhabits the same plane of existence as Porsche's legendary 959. Better driving through science. Of course, the Nissan GTR 2010 Modification is faster, more nimble and worlds less expensive than my favorite Porsche. Now I'm not blind, deaf or numb. A certain number of imperfections do add flavor. But that's hardly the only way to build character. Might I suggest a 485-hp, twin-turbo 3.8-liter V6 coupled to a fancy-pants AWD system and a dual-clutch tranny from hell? For my money, that's about as soulful as a car gets.

1987 Yamaha FZX 750 - Motorcycle Touring


1987 Yamaha FZX 750 - Motorcycle Touring. Yamaha FZX 750 Wallpaper, Yamaha FZX 750Posters, Yamaha FZX 750 Pictures.

1987 Yamaha FZX 750 - Motorcycle Touring


1987 Yamaha FZX 750 - Motorcycle Touring. Yamaha FZX 750 Wallpaper, Yamaha FZX 750Posters, Yamaha FZX 750 Pictures.

Saturday, January 30, 2010

Suzuki Thunder 250 Modification

Suzuki Thunder 250 Modification - Custom Suzuki Thunder. Motorcycle Touring

All motor design modifications here. Like yamaha R6, Yamaha Vixion, yamaha scorpio, yamaha FZ, yamaha R1, yamaha mio, suzuki thunder, suzuki satria, honda tiger, honda CBR etc.


Motorcycle head lamp variations

Thunder 250 specification modifications are:

KALIPER front: Brembo 4 piston Brembo master
KALIPER back: Nissin 2 piston Nissin brake master
Front disc: 300 mm
Cakarm back: 240 mm
Sok fronts: GSXR400 Showa
Sok back: Showa
Swing-arm: Pro-arm VFR 400
Footstep: Copy of the Honda CBR400
Front rim: Enkei 3:50 x17
Front tires: Pireli 120/60-17
Rear rim: Enkei 4:50 x18
Rear tires: Pireli 160/60-17
Handlebar: Kawasaki Ninja, Yamaha FZR Raiser
Closed tank: Kawak Ninja
Deltaboks: Fiberglass
Fairing: Acerbis
Speedometer: Suzuki Bandit
Headlights: Acerbis
Windshield: Acerbis
Body rear: JMS Design
Stirrup back: Suzuki Thunder
Taillights: Honda CBR150
Hood: JMS Design


Modification of the single arm Suzuki Thunder

Suzuki Thunder 250 Modification

Suzuki Thunder 250 Modification - Custom Suzuki Thunder. Motorcycle Touring

All motor design modifications here. Like yamaha R6, Yamaha Vixion, yamaha scorpio, yamaha FZ, yamaha R1, yamaha mio, suzuki thunder, suzuki satria, honda tiger, honda CBR etc.


Motorcycle head lamp variations

Thunder 250 specification modifications are:

KALIPER front: Brembo 4 piston Brembo master
KALIPER back: Nissin 2 piston Nissin brake master
Front disc: 300 mm
Cakarm back: 240 mm
Sok fronts: GSXR400 Showa
Sok back: Showa
Swing-arm: Pro-arm VFR 400
Footstep: Copy of the Honda CBR400
Front rim: Enkei 3:50 x17
Front tires: Pireli 120/60-17
Rear rim: Enkei 4:50 x18
Rear tires: Pireli 160/60-17
Handlebar: Kawasaki Ninja, Yamaha FZR Raiser
Closed tank: Kawak Ninja
Deltaboks: Fiberglass
Fairing: Acerbis
Speedometer: Suzuki Bandit
Headlights: Acerbis
Windshield: Acerbis
Body rear: JMS Design
Stirrup back: Suzuki Thunder
Taillights: Honda CBR150
Hood: JMS Design


Modification of the single arm Suzuki Thunder

Friday, January 29, 2010

Modification Suzuki Thunder Like Aprilia RS 125


Modification Suzuki Thunder Like Aprilia RS 125

Suzuki Thunder 125cc lansiran 2004 this was already out of shape as the original form. With the use of waste legs APRILIA RS 125 can change the look of the original Thundie suzie be the alias of the feminine to masculine. According to the owner, thunder weakness in the sector has a very visible foot small or virtually all forms that are not proportional, "was plump bony legs".



The choices are the feet of APRILIA RS125. besides the form of a futuristic and very luxurious look, this usage there is also another reason that is "light". Due to the foot of this cesspool was using aluminum alloy materials as base materials. Unlike the feet of local production of the majority still use the iron as a material. This usage does not automatically make the Thunder, which caused has only a relatively small cc. This result could thunder run there here, chatter is now a part of the family in Jakarta Thunder Motor Club (JTMC).

Use of any tire that is already fairly large 140 x 70 x17 "to measure the back with a bandage made in the name of Dunlop Arrowmax sakura country. Meanwhile, the front tires wrapped wheel size 110 x 80 x 17 ". But the use of feet is pretty big. A little help with the replacement of the Pilot & mainjet racing and a racing clutch and canvas with RMG brand, this change is felt by rider enough, which is also supported by the front brakes are already adjustable



While self-telescopic front has not been changed again, but with upside down Aprilia too. With the front discs have not been on the left again but moving on the right. Trus the question arises, how to read the speedometer thunder, if the disc had changed places, the automatic location of teeth follow-up nanasnya changed?. For this case the owner had to replace the reader system speed (speedometer) with digital rather than analog or needle system. By changing the digital speedometer which happened to use Speedo from Suzuki Satria F 150, also changed the location of reading. Not anymore with pineapple teeth in the front wheel speed sensor but placed in the front gear. This bike looks so futuristic and sophisticated added.

Meanwhile, in terms of clothing, this thundie not too many changes, only the headlamp which has been replaced with a headlamp also satria F150, Aprilia spakboard Diablo, airscope tiger and custom fiber airscope engines, and equally important is mounted behind breket order to carry while touring. Breket installation also has a reason, in addition to covering the peculiarities of the hind legs longer than the body and also supports the concept of a themed motorcycle Street and touring fighter, which aims to come on the streets and taken in as well touring monggo invited.

This manufacturing process takes about 2 months and ate a pretty good budget but was carried off her satisfaction with the success of this bike as a motorcycle on display at the Suzuki stand at Pekan Raya Jakarta (PRJ). Want to see the look of this RS125 Suzuki Thunder on stage




Modification Suzuki Thunder Like Aprilia RS 125


Modification Suzuki Thunder Like Aprilia RS 125

Suzuki Thunder 125cc lansiran 2004 this was already out of shape as the original form. With the use of waste legs APRILIA RS 125 can change the look of the original Thundie suzie be the alias of the feminine to masculine. According to the owner, thunder weakness in the sector has a very visible foot small or virtually all forms that are not proportional, "was plump bony legs".



The choices are the feet of APRILIA RS125. besides the form of a futuristic and very luxurious look, this usage there is also another reason that is "light". Due to the foot of this cesspool was using aluminum alloy materials as base materials. Unlike the feet of local production of the majority still use the iron as a material. This usage does not automatically make the Thunder, which caused has only a relatively small cc. This result could thunder run there here, chatter is now a part of the family in Jakarta Thunder Motor Club (JTMC).

Use of any tire that is already fairly large 140 x 70 x17 "to measure the back with a bandage made in the name of Dunlop Arrowmax sakura country. Meanwhile, the front tires wrapped wheel size 110 x 80 x 17 ". But the use of feet is pretty big. A little help with the replacement of the Pilot & mainjet racing and a racing clutch and canvas with RMG brand, this change is felt by rider enough, which is also supported by the front brakes are already adjustable



While self-telescopic front has not been changed again, but with upside down Aprilia too. With the front discs have not been on the left again but moving on the right. Trus the question arises, how to read the speedometer thunder, if the disc had changed places, the automatic location of teeth follow-up nanasnya changed?. For this case the owner had to replace the reader system speed (speedometer) with digital rather than analog or needle system. By changing the digital speedometer which happened to use Speedo from Suzuki Satria F 150, also changed the location of reading. Not anymore with pineapple teeth in the front wheel speed sensor but placed in the front gear. This bike looks so futuristic and sophisticated added.

Meanwhile, in terms of clothing, this thundie not too many changes, only the headlamp which has been replaced with a headlamp also satria F150, Aprilia spakboard Diablo, airscope tiger and custom fiber airscope engines, and equally important is mounted behind breket order to carry while touring. Breket installation also has a reason, in addition to covering the peculiarities of the hind legs longer than the body and also supports the concept of a themed motorcycle Street and touring fighter, which aims to come on the streets and taken in as well touring monggo invited.

This manufacturing process takes about 2 months and ate a pretty good budget but was carried off her satisfaction with the success of this bike as a motorcycle on display at the Suzuki stand at Pekan Raya Jakarta (PRJ). Want to see the look of this RS125 Suzuki Thunder on stage




Thursday, January 28, 2010

Find The Top Auto Insurance Company

Before investing in insurance for your car, it is important to gather the facts car insurance to help you make the right decisions about policy and the insurance company is best for you. Are you looking in the ever popular Saga car insurance, car insurance or travel to other companies or auto insurance for teenagers, here are some things you should know for this process.

With the rising cost of insurance today, more and more willing to find a cheaper policy for their car. Although it is possible to find a good rate for your car. Cheap car insurance can be found by a number of options. Here are some facts from the car insurance that will play an important role in the amount you pay for insurance.
Type of car you drive will make a big difference. Sports cars such as Ferrari, Corvette, etc. all cost more than the typical economy car because the type of driver who buy them. Also, your driving history will play a major role. The best records you have, the less you pay.

Furthermore, by installing safety and anti-theft devices in your car, you may be paying a lower premium for car insurance. This car is less than the risk of theft, making them much more attractive to insurance. Also, there are some decisions that you can do to reduce your expenses as well. For example, many companies give discounts or more than one policy under them.

In addition, sometimes you can often find cheaper auto insurance exclusively with companies online that you can in the offline world. By doing thorough research, you can quickly compare quotes from several companies and save you hassle calling everyone by phone.

But, really sure that you compare the features included in the policy. For example, a company may appear cheaper than others, but in fact, ignoring some key elements you need for your insurance. Cheap is not always better, especially in motor insurance.

Policies differ greatly in features and functionality, and many companies will offer incentives to jump on board with them. This is often at this point that you can really cash in some big savings when the AUTO insurance contract right. Remember, never buy just because a company is cheap, take a glance at the features they offer, especially their overall reputation and reliability.First, you must determine whether you need a third party or comprehensive insurance. Clearly, this decision will depend on many factors such as age, type of car you drive, old cars, etc.

In addition, there are many other options that could be included in your car insurance that does not come standard. Some of these options may include the obligation to repair the damage insurance and medical coverage. Some companies will include this standard option, and some of your extra. Therefore, make sure you shop around before buying.
Here's the bottom line: there are many car insurance companies out there today. Although this article does not suggest a specific person, the most important thing, as always, is to do your homework and gather as many facts auto insurance policies as possible before deciding which is right for you. Only once you have done this you should purchase an insurance policy for your car.

VARIO MODIFICATION NEW INNOVATION

inovation modified motor was born in 2009 from Madiun, East Java. Donny is Dwi Budianto of the 73 Motor penggagas. Honda Vario 2007 not own dirombak steering wheel with memelarkan alias with a low rider car velg. Honda Skubek jig that can increase body alias-down such as the withdrawal of many done in the car modif.

Front monoshock
Indeed, applying the concept MEFRIK Donny aka Modern, Estetika, Functional, rational, Innovation, and Creative skubek honda vario. Most prominent and can be spelled breakthrough modif 2009 Suspensi namely the air (air-sus), which can increase body make-down, as many modifications adopted in the car in the country."This is as big matic trends in the Japanese," says Donny. To operate the water-sus, it uses bertekanan compressor 550 psi. To use Shockbreaker hidraulik berdiameter 4 cm are usually used on the car door.

Because funsgi shock only to ride-down body, then add Donny shock placed under the saddle to the front of the stick near the shield. Function, in addition to set the compress and rebound, but also to stabilize the body. To operate the water-sus, using two buttons placed on the stabilizer.Innovation from Donny, note the shock front. He adopted the monoshock. "His own use, such as pipes and model in the Vespa," he said. So that custom look, the fiber was wrapped and given the chrome.

Not only that. Try habituate lamp-light. Future use mica lamp Honda Airblade and have a surplus, in part at the custom-life model of projector light car. Meanwhile, the dusk is mengaplikasi LED.

The creation of the rear motor 73. Of the tapering stern line with all-LED lights. There are also lights and brake sein.

Satria F 150cc Modif 2010

SUZUKI SATRIA 150 F EXTREME MODIFICATION
You would not anticipate that a adapted motorcycle belonged to a 55-year-old grandfather. His name Suryana and breeze modif rombakan after-effects are to his satisfaction. Mean, bodies are afterward the actual Jakarta developments and trends modifications.It was so, the abject motor victim Suryana attraction is affectionate of a avoid from Suzuki Satria F-150. To change the "sex" from the avoid into "male model", he handed over the avoid to Tauco Custom (TC) in the Sudirman, South Jakarta, is accepted for authoritative such changes.

SUZUKI SATRIA 150 F EXTREME
full striping modification

suzuki satria fu full striping
STRIPING SUZUKI SATRIA FU Related Posts with thumbnails for bloggerblogger widgets
SUZUKI SATRIA 150 F Rims
Topo Goedhel Atmodjo, skipper TC, annihilate the motor to chase his grandfathering posture. Therefore, the motor was fabricated not so aerial that the accepted Monoshock still in use, including the aboriginal beat arm is alone in-custom bit. Meanwhile, the advanced abeyance upside bottomward archetypal that he fabricated to break adjusted. Gas catchbasin was additionally accountable to accommodate 6 liters. Display motor looks solid because there is a DELTABOX of aqueduct 1 / 2 inches.



satria f 150 cc 2010


Technology FU150 that had engine 147cc, 4 not, DOHC 4 valves, water cooled with SACS (with Suzuki Advanced Cooling System), this that pushed me to buy

SUZUKI SPIN MODIFICATION

Modifications suzuki spin this is the year 2009 with the concept modif x-treme where modifications in the contest, suzuki spin must in the form of modifications have a very good and the jury could draw attention to the modifications in the contest. suzuki spin on the modifications in the form of a very different from the others because matic motor suzuki spin have the form of a unique and different and it is a difficulty in making the modifikator draft modifications suzuki spin them.
MODIFIKASI SUZUKI SPIN 2009Spin Modification Full Airbrush
MODIFIKASI SUZUKI SPIN 2009

MODIFIED SPIN COLUMN FOR SIMPLE AND RAPID PLASMID DNA EXTRACTION

Modified Spin Column for Simple and Rapid Plasmid DNA Extraction

Cross-Reference to Related Applications

This application claims priority to United States provisional patent application number 60/941,032 filed 31 May 2007; the disclosure of which is incorporated herein by reference in it entirety.

Field of the Invention

This invention relates to an improved system and method for nucleic acid purification. More specifically, it relates to a simple and rapid system and method for the extraction and purification of plasmid DNA from cells.

Background of the Invention spin

foto modifikasi motor spin

Plasmids are double-stranded supercoiled DNA molecules that range in size from 1 kb to more than 200 kb. Plasmids are useful tools in genetic engineering. They are widely used as vectors to carry foreign DNA; such that the foreign DNA is amplified and isolated or expressed. Plasmid DNA has also been utilized in the development of vaccines and in gene therapy.

The analysis and in vitro manipulation of plasmid DNA is typically preceded by an isolation step in order to free the nucleic acid from unwanted cellular contaminants which may interfere with subsequent processing procedures. A mini-scale sample preparation from an overnight bacterial culture of 1-5 ml generates more than enough plasmid DNA (~ few micrograms) for many of these applications.

The most common plasmid DNA extraction protocols exploit reagents originally developed by Birnboim and DoIy (Birnboim, H. C. and DoIy, J., Nucl. Acids Res. 7, 1513

(1979)), to separate supercoiled plasmid DNA from bacterial genomic DNA, RNA and protein. These reagents, developed many years ago, work on the principle of sequential use of three buffers, commonly referred to as buffer I, II and III. They each have distinct compositions to bring about plasmid enrichment and separation from contaminants. Buffer I is used to resuspend the bacterial pellet obtained by an initial centrifugation step of an appropriate bacterial culture. Once resuspended, buffer II is added which contains SDS detergent and NaOH. These components lyse the bacteria and denature the genomic DNA (pH>12). Buffer III typically contains a chaotrope to further denature protein, the chaotrope also promotes binding of plasmid DNA to the silica matrix commonly used in spin columns. Buffer III also usually contains potassium acetate to rapidly neutralize the combined solutions. The addition of buffer III causes contaminants to "crash-out" of solution owing to the formation of insoluble complexes driven by rapid re-naturation of genomic DNA and the potassium salt of the detergent.

spin modif

The insoluble flocculant material has traditionally been removed by a centrifugation step to "pack" the flocculant material at the bottom and side of a centrifugation tube (See, e.g. ILLUSTRA™ plasmidPrep Mini Spin Kit, GE Healthcare, Piscataway, New Jersey). The clarified plasmid-containing solution is subjected to a chromatographic separation. For mini-scale purification, the clarified solution is usually applied to a minispin column containing a glass fiber matrix or silica membrane. Plasmid DNA binds to the column in the presence of a chaotrope, while soluble impurities do not bind. After the soluble impurities are washed off, the plasmid DNA are eluted with an appropriate elution buffer.

Recently, alternative buffer compositions have been developed for plasmid DNA extractions based upon 96-well plates, which minimizes the formation of flocculants during bacterial lysis (DIRECTPREP™ 96 Miniprep Kit, Qiagen Inc., Valencia,

California). The use of these buffers generates little precipitated cellular components and eliminates the need to remove the flocculants before column loading of the DIRECTPREP™ 96-well plates. A 96-well plate pre-fϊlter is used to capture any residual precipitants from clogging the silica membrane. However, the columns may become clogged if the cell density is high.

It is advantageous to further simplify the process therefore to provide a more efficient plasmid DNA purification method.

Summary of the Invention In general, the instant invention provides improved methods, systems and kits for rapid isolation of plasmid DNA from plasmid containing cells.

In one aspect, the invention features a method for the rapid isolation of plasmid DNA, including: a) collecting plasmid-containing cells and resuspending them in an aqueous buffer; b) incubating the resultant mixture with a lysis/denaturation solution to lyse the cells and denature DNA; c) neutralizing the mixture with a renaturation solution to generate a renatured mixture of dissolved plasmid DNA and flocculants containing insoluble genomic DNA and cellular debris; d) loading the renatured mixture directly to a modified spin column without first removing the flocculants from the mixture, which column having a pre-filtration disc (e.g., pre-filter) on top of a matrix; e) passing loaded sample mixture through the column such that the flocculants are packed on top of the pre- filtration disc while plasmid DNA binds to column matrix; f) washing the column with a wash solution to remove soluble impurities; and g) eluting plasmid DNA from the column with an elution buffer. It has been found surprisingly that by introducing an integral pre- filtration disc, there is no longer a need to remove flocculants containing cellular debris prior to loading the spin column. Instead, the flocculants stay on top of the pre-filtration

disc throughout the purification process and do not interfere with subsequent wash or elution of the plasmid DNA.

modifikasi suzuki spin

In a second aspect, the invention provides a modified spin column for the rapid isolation of plasmid DNA from plasmid-containing cells, comprising: a matrix; a support filter underneath the matrix; a pre-filter on top of the matrix; and a housing for the matrix and filters. Preferably, the modified spin column contains a glass fiber matrix and the pre- filter and support filters are made of porous sintered polyethylene. In a variation of the modified spin column, a depth filter is included between the separation matrix and the pre-filter to further enhance the performance of the system. In another aspect, the invention provides kits for rapidly isolating plasmid DNA, including the modification spin column, reagents and user manual.

Certain aspects of the invention allow simultaneous isolation of a large number of different plasmids. The spin modif columns can be joined together to take the form of a microtiter plate. By this kind of an arrangement, a number of different plasmid containing cell cultures can be processed simultaneously. It is noted that all centrifugation steps can be replaced with vacuum.

The above and further features and advantages of the instant invention will become clearer from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 shows a schematic diagram of the spin modif column according to one embodiment of the invention.

Figure 2 shows a gel image of four samples prepared according to one example of the invention, before (left) and after (right) a HindIII digest. 400 ng of DNA was used for each digest, with 1 unit of HindIII, and incubated at 370C for 2 hours. Far left: markers.

Figure 3 shows a schematic diagram of the modified spin column according to a variation of one embodiments of the invention. A depth filter is included between the pre-filter and the main separation matrix.

Figure 4 shows a gel image of plasmid DNA isolated using the modified combination pre- filter/depth filter systems according to the scheme of Figure 3. The numbers represent either controls or a particular combination according to Table 1.

Detailed Description of the Invention

The invention features improved processes, systems and kits for rapidly isolating plasmid DNA from plasmid containing cells, in particular for downstream applications in molecular biological research, such as cloning and sequencing. As used herein, the term "plasmid" refers to supercoiled DNA molecules (single or double stranded) that are maintained in a host cell separate from the host cell genome. Plasmids can be of a high copy number or low copy number and can carry any gene or external piece of DNA, either genomic or synthetic, encoding protein or peptide of interest, from any source.

In general, the improved process for isolating plasmid DNA includes modification of a spin column such that it eliminates the need to remove the insoluble flocculant cellular debris generated from the lysis of the cells, before loading the column, this simplifies the work flow and shortens the protocol significantly. A spin column for plasmid DNA isolation generally contains a separation matrix placed on and supported

physically by a disc of porous material more commonly referred to as a frit, typically made of sintered polyethylene. The holes so formed during the production of the frit material allow the unhindered passage of aqueous solutions and more importantly aqueous solutions containing plasmid DNA. The frit material is inert and does not interact to any great extent with DNA. The separation matrix is preferably a glass fiber matrix or a silica membrane. Alternatively, the matrix is a zeolite, or an organic matrix such as a resin or polymer.

One embodiment of the invention includes a modification of the spin column with the addition of an integral pre-filter on top of the separation matrix. One example of a pre-filter is a porous sintered polyethylene or polypropylene, similar to the support frit underneath the separation matrix.

The use of the pre-filtration disc does not have to be the same composition as the lower supporting frit and indeed an optimal column might be composed of alternative materials having different filtration/binding characteristics. A thinner "pre-filter" material (e.g., cellulose absorbent paper or polypropylene mesh) may allow improved assembly of the column where sheets of appropriate components are layered together prior to die- cutting and positioning within a column moulding. Optionally, an O-ring can be used to secure the "pre-filter" (Figure 1).

A variation of the embodiment additionally includes a depth filter between the pre-filter and the separation matrix (Figure 3). A combination of both a pre-filter and a depth filter further reduces residual contaminant flow-through from the pre-filter, thus is preferable for certain applications. A preferred depth filter is one that captures any residual flow-through contaminants from the pre-filter yet does not retain plasmid DNA during elution. A suitable depth filter is a glass microfiber filter.

Plasmids isolated in accordance with the invention can be of any origin. Most commonly, microorganisms like bacteria, such as Escherichia coli (E. coli), are used for culturing the plasmids, but the use of host cells is not limited and can be prokaryotic or eukaryotic cells. The host cells harboring the plasmid can be cultivated in a number of ways well known in the art, e.g. in incubator, bioreactor, fermentor etc. The plasmid isolated according to the invention can be of virtually any size, e.g. in the range of about 1 kb up to about 20 kb. As an upper limit, the isolation of cosmids and artificial chromosomes is also encompassed, the size of which may be up to about 50 kb and 500 kb, respectively. The modified spin column is suitable for extraction of plasmid DNA from standard cultures of bacteria. The inclusion of a pre-filter within a spin column eliminates the need to remove flocculant material generated by alkaline lysis, prior to addition of sample to the DNA-binding column. This modified column is especially suited for the so called miniprep of plasmid DNA from 1-5 ml overnight culture. For a miniprep, traditionally, lysate is clarified by a 5-10 minute spin in a micro-centrifuge before addition of the clarified lysate to the microspin column. Using the modified device, two steps are removed from the process without affecting quality of isolated product. Purification of plasmid DNA with the modified device can now be done in 6-8 minutes for a miniprep, compared to traditional process which typically takes about 20 minutes to complete.

The modified spin column is also suited for the preparation of plasmid DNA in a larger scale. For example, between 10-50 ml overnight culture could be used as a starting material, and larger spin columns are devised to accommodate the increased volume of the lysate. A modified, larger column with an integral prefilter achieves similar benefits as a modified microspin column.

During the experimentation it was found that the use of the pre-filter modified microspin column in combination with a fixed-angle microcentrifuge enabled the insoluble flocculent material to be pelleted "over to one side" so that occlusion of the frit pre-filter was less likely to occur. Even without a fixed-angle rotor, using a vacuum that distributes flocculant material across the entire surface of the frit, good quality plasmid DNA is still obtained that can be digested and sequenced.

By deploying the modified spin column, it has been possible to achieve multiple benefits. First, it enables the addition of lysed sample to the modified column without removal of flocculants (pre-processing). It also ensures total utilization of sample without incurring transfer losses owing to pre-processing. It further provides an improvement in the ease of use and time for completion, speeding up the process by more than 50%. The introduction of a pre-filter also stabilizes the separation matrix, known to be fragile and liable to partial fragmentation.

Methods for isolating plasmid DNA generally starts from culturing the host cells containing the plasmid. When the culture is ready, the cells are recovered by e.g. centrifugation or filtration. The cells can be stored, for example in a freezer, or processed immediately. The process for isolating plasmid DNA includes first collecting plasmid- containing cells and resuspending them in an aqueous buffer; then incubating with a lysis/denaturation solution to lyse the cells and denature DNA; followed by neutralizing the mixture with a renaturation solution to generate a renatured mixture of dissolved plasmid DNA and flocculants containing insoluble genomic DNA and cellular debris. In one aspect, the improved method includes loading the renatured solution with the flocculants directly to a modified spin column having an integral pre-filtration disc (pre- filter) on top of the separation matrix. The solution is then passed through the modified spin column by centrifugation or vacuum, such that the flocculants are packed on top of

the pre-filtration disc while plasmid DNA binds to separation matrix. The modified spin column is washed with a wash solution to remove soluble impurities; and plasmid DNA is eluted from the column with an elution buffer. It is surprisingly discovered that although the flocculants remain packed on top of the pre-filter during the washing and elution steps, high quality plasmid DNA is isolated that is suitable for subsequent molecular biology analysis.

The protocols for cell lysis and denaturation of cellular debris are well known. A particularly useful aqueous buffer for resuspending plasmid-containing cells contains an isotonic buffer (e.g. a Tris buffer; or a sucrose or glucose solution), a chelating agent (e.g. ethylenediaminetetraacetic acid (EDTA) or (CDTA)) and an RNAse. This buffer may also optionally include lysozyme to further weaken cell walls. After the cells are resuspended, the cells are lysed and linear DNA is denatured, preferably by incubation in an alkaline lysis solution. Thorough lysis and denaturation can be accomplished by mixing the resuspended cells with a sodium hydroxide, sodium dodecyl sulfate solution. A third, renaturation solution (e.g. an acetate buffered solution, containing a chaotropic salt) is then added to yield a mixture containing plasmid DNA, insoluble clots of linear DNA and cellular debris.

According to one aspect of the invention, the renatured mixture of dissolved plasmid DNA and insoluble flocculants are loaded to the modified spin column. Through vacuum or centrifugation, liquids in the mixture passes through the column, leaving on top of the pre-filter a packed layer of flocculants, in the meantime plasmid DNA binds to column matrix. A wash solution is then applied to remove soluble impurities; and plasmid DNA is then eluted from the modified spin column with an elution buffer. The flocculants remain packed on top of the pre-filtration disc during the washing and eluting steps but does not affect the quality of the plasmid DNA isolated.

The addition of a depth filter between the pre-filter and the separation matrix results in slightly better quality DNA. Thus it is preferable to include a depth filter in the modified spin column for certain preparations. The workflow, however, does not change from the protocol which includes the pre-filter only. Certain aspects of the invention allow simultaneous isolation of a large number of different plasmids. The modified spin columns can be joined together to take the form of a microtiter plate. Especially preferred are microtiter plates in the 96 well format. By this kind of an arrangement, a large number of plasmid containing cultures can be processed simultaneously. It is noted that all centrifugation steps can be replaced with vacuum.

Examples

The following examples serve to illustrate the plasmid DNA purification processes according to embodiments of the present invention and are not intended to be limiting.

1. The protocol

The protocol is suitable for the rapid extraction and purification of plasmid DNA from 1.5 ml cultures of E. coli. The procedure can be completed in less than 10 minutes to yield plasmid DNA with a purity and quality compatible with many common molecular biology techniques, including cloning, restriction enzyme digestion, PCR amplification and DNA sequencing.

The plasmid DNA yield from a freshly grown E. coli strain containing a high copy number plasmid (>300 copies/cell) and grown to A60O approximately 2.5 is typically 4 to

The protocol utilizes a simple plasmid DNA purification process, employing a modified alkaline cell lysis procedure and a silica-based membrane. No organic solvents are used; instead, chaotropic salts are included to denature protein components and promote the selective binding of plasmid DNA to the silica membrane. Denatured insoluble contaminants are retained on top of pre-filter, while soluble contaminants are easily removed by subsequent washing. The purified plasmid DNA is eluted in a low ionic strength buffer, at a plasmid concentration suitable for most molecular biological applications.

The following provides a step by step protocol: 1. Transfer 1.5 ml from a fresh overnight culture to a microcentrifuge tube. To pellet bacteria, centrifuge (13 000 x g) for 30 seconds. Discard supernatant and re-centrifuge. Remove any residual supernatant using a pipette.

2. Thoroughly resuspend the pellet by adding 150 μl lysis buffer (10OmM Tris- HCl pH7.5; 1OmM EDTA; 0.2mg/ml RNase A), and either vortexing, pipetting up and down or scraping the base of the microcentrifuge tube across the surface of an empty pipette tip rack.

3. Cell lysis - Add 150 μl lysis buffer (20OmM NaOH; 1% SDS) and mix immediately by gentle inversion (approximately 5 times) until solution becomes clear and viscous. 4. Neutralisation - Add 300 μl neutralization buffer (4.4M Guanidine HCl,

0.65M potassium Acetate and 3.1M Glacial Acetic Acid), and mix immediately by gentle inversion until the precipitate is evenly dispersed. 5. Transfer the neutralized mixture to the modified microspin column

(approximately 600 μl). Close the lid of the column gently. Centrifuge (13

000 x g) for 30 seconds. Discard the flow through by emptying the collection tube.

6. Wash the column with 600 μl wash buffer (2mM Tris-HCl pH8; 0.2mM EDTA and 80% ethanol) and centrifuge (13 000 x g) for 30 seconds. Discard the flow-through and repeat the wash one more time with a 60 second spin.

7. Move the modified microspin column into a fresh microcentrifuge tube and add 100 μl elution buffer (1OmM Tris-HCl pH8) directly onto the centre of the column. Incubate the column for 30 seconds at room temperature. Microcentrifuge (2 000 x g) for 60 seconds to recover the plasmid DNA as flow through in the microcentrifuge tube.

Purified plasmid DNA concentration should be determined by UV spectrophotometry (A260) and through comparison with a known standard by agarose gel electrophoresis and subsequent densitometric analysis. If available, the UV spectrophotometric ratios A26o:A28o and A26o:A23o provide a limited indication of purity as measures of protein and salt contamination.

2. Purification of plasmid DNA using a modified microspin column containing a prefilter

Overnight cultures of E. coli TOPlO transformed with pCORON1002-EGFP-Cl were processed following the protocol described above. Four individual cultures were prepared and plasmid DNA was isolated according to the protocol. Modified microspin columns contained a pre-filtration disc of a porous, sintered polyethylene. The samples had a mean yield of 6.7 μg. The plasmids are suitable for downstream molecular biology applications as illustrated by restriction enzyme digestion (Figure T).

3. Purification of plasmid DNA using a variation of the modified microspin column

To further reduce extraction time whilst maintaining the purity/quality of the isolated DNA to a level comparable to that generated using traditional microspin systems, the inclusion of a depth column between the pre-fϊlter and the main separation matrix was tested (Figure 3).

The above protocol was used for plasmid DNA isolation, with slight modification. Briefly, 125 μl re-suspension buffer and lysis buffer, respectively, was used for each culture, while 250 μl neutralization buffer was used. Crude lysate was added directly onto the integral filtration/plasmid DNA binding column and centrifuged at 13,000 g for 60s in a microcentrifuge. The columns were washed twice with 400 μl wash buffer before DNA elution. Absorbance data was determined using a Nanodrop NDlOOO spectrophotometer.

A number of pre-filter and depth filter combinations were tested, using a silica membrane column as the main plasmid DNA binding matrix (the column from ILLUSTRA™ plasmidPrep Mini Spin kit). To compare the quality and yield with traditional protocols, controls were included. The control experiments were performed following manufacturer's protocols, except the pre-filter only control which was performed following the current protocol. The depth filter used was the Whatman GF/B glass microfibre depth filter. Table 1 lists the pre-filters tested in combination with the Whatman GF/B glass microfibre depth filter, and the control experiments performed.

Table 1 : Summary of pre-filter/depth filter combinations tested.

For each pre-fϊlter/depth filter combination (or control experiment), at least three parallel experiments were run. It was found that with an integral pre-filter/depth filter combination, the time needed to complete a plasmid DNA isolation experiment was about 7.5 min. In comparison, the ILLUSTRA™ plasmidPrep Mini Spin kit took about 9 min to complete, while the QIAPREP™ Spin Mini kit took about 19 min to complete. In general, the modified system with both a pre-filter and a depth filter generated comparable amount of plasmid DNA as the control extractions irrespective of the material used as the pre- filter.

The quality of the isolated plasmids were also comparable to the ones isolated using the control kits. Low level of protein contamination was observed. The amount of particulates in the final elution was also comparable to control extractions. Salt levels were lower than the control QIAPREP™ or ILLUSTRA™ plasmidPrep kit. The majority of native plasmid DNA was in the supercoiled configuration (Figure 4; 300 ng of DNA loaded on 1% agarose gel)). Therefore in general the quality of the isolated plasmid DNA was comparable to control extractions.

The modified microspin column with both a pre-filter and a depth filter combines the speed of a pre-filter only system (i.e. 7.5min) with quality associated with traditional spin extraction methods/kits. Even though the Whatman GF/B micro-fibre depth filter probably binds some plasmid DNA in the presence of the chaotrope, in an integral filter format the plasmid DNA can be recovered during the final elution step.

All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.

 
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