Virtually everything from the desktop interface has made its way to mobile, and form-filling is now supported on both iOS and Android, making for a much more complete experience.īoth: Android (Image credit: Tom's Guide) RoboForm has massively overhauled its mobile apps since the last time I reviewed them, and the apps now have a clean, modern look. RoboForm claims Zxcvbn offers a more accurate depiction of how hard it would be to crack a given password. This is different from the quotas for lowercase letters, uppercase letters, digits and symbols (LUDS) that most password-strength estimators rely on. RoboForm calculates password strength using the open-source tool Zxcvbn, which detracts points for dictionary entries, common names and common passwords. Next to every compromised password, RoboForm provides a link to the website in question so that you can quickly log in and change the password. In May 2021, it began importing lists of compromised passwords from Have I Been Pwned, a non-profit online tracker of passwords and email addresses exposed in data breaches. It audits the security level of your passwords and singles out any reused passwords. Emergency Access was at one time obscured in the RoboForm interface, with the menu bar being the only way to access it, but has since been brought into the browser extensions and been added to the primary navigation bar. While the use case for Emergency Access is if you are either incapacitated or dead, it is also a potential lifesaver should you forget your master password. The functionality is largely the same among the four, which is an improvement the last time I reviewed RoboForm, the website was a read-only experience. RoboForm gives you four different ways to access your account on the desktop: the standalone app, the browser extension, the menu bar and the website. It will also import from any browser that supports the RoboForm extension on that platform, including on Chrome OS and Linux.įinally, you can install RoboForm on the rest of your devices and your data will immediately sync between them as soon as you enter your RoboForm master password. RoboForm supports imports from 1Password, Dashlane, LastPass, NIS, Xmarks, KeePass, Keeper, Enpass, Password Boss, Zoho Vault, True Key and Sticky Password, as well as generic CSV files. If you were already using some other method of password management, either in your browser or with another stand-alone password manager, you should import your data. As with most password managers, there is no way to recover this master password if you lose it. RoboForm requires that the master password be at least 8 characters long with a minimum of 4 non-numeric characters. Now you need to create your account, which requires just an email address and a master password. The universal installer for macOS and Windows will also install the full-featured browser extensions for all supported browsers. It will recognize the device that you are browsing from and recommend the correct version. To get started, just navigate to the RoboForm downloads page. Google Chrome was my primary browser across all platforms but testing on macOS and iOS was also done with Safari. Chrome OS is supported via the Google Chrome browser extension.įor this review, I used RoboForm on a 2017 MacBook Pro 15 running Windows 10 and macOS 10.14 Mojave, an iPhone 7 Plus, and a Google Pixel 3. Linux users have standalone extensions available for Mozilla Firefox and Google Chrome. RoboForm offers similarly extensive support on mobile, going all the way back to iOS 13 and Android devices running Android 5 Lollipop. Mac users must be running macOS 10.13 High Sierra or later, and the Mac installer includes browser extensions for Apple Safari, Google Chrome and Mozilla Firefox. The Windows universal installer will add browser extensions to Microsoft Internet Explorer, Mozilla Firefox, Google Chrome, Microsoft Edge, Opera and Windows applications, and updated versions of the browser extensions were rolled out in March 2021. Support for Windows goes all the way back to Vista.
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Remote Data disk storage is billed separately from virtual machines. Dsv4-series VMs feature Intel Hyper-Threading Technology. it makes validation a bit trickier but I agree it's worthwhile. The Dv4-series sizes offer a combination of vCPU, memory and remote storage options for most production workloads. Tl dr: unlikely before it's out of preview on the compute side, but probably sooner or later. A better example might be Azure/kubernetes-volume-drivers#82, but fortunately I don't think that's very standard behavior. I'm mostly worried about the intersection with other features which may use the temp disk like #1753 (although admittedly, ephemeral os on cache disk would solve the same root problem that moving docker root to temp solves). I don't think we will add cache disk before it's out of preview, but we will probably support it at some point after that. I wrestled with this exact issue when looking at it the first time, there's an unfortunately large matrix of various skus supporting things (ephemeral os, cache size, temp disk available/size, requested os disk size), which we have to consider/expose to end users. For me this is a better solution as it offers me larger disks on smaller VM sizes. My understanding is that you see Standard_HDD for ephemeral OS because that's the storage class of the "clean" copy (admittedly not my product area, so don't quote me on that □).ĭo you know if you'll be supporting Ephemeral OS Disks on temp disks in AKS soon as well? I noticed that was in Preview. With non-ephemeral OS, the diff still writes back to Azure storage persistently. With ephemeral OS, the diff disk is exclusively local to the VM and will not persist data after e.g. When you provision the VM, you also need a diff disk to write changes made during runtime. To really get in the weeds, my understanding is you always need a "clean" copy of the image to do operations like reimage. Standard HDD showing in disks is just a subsequent artifact and is not indicative of the actual local storage device that is enabling Ephemeral OS via the cache. With the arrival of these publications, a new trend of village studies was ushered in Indian sociology. Dube authored the full-length monograph on a village in Andhra Pradesh under the title, Indian Village. Srinivas), and Village India (edited by Mckim Marriott). Majumdar), India’s Villages (edited by M.N. The anthologies were Rural Profiles (edited by D.N. In 1955, four major publications came out, three of which were anthologies of articles written by social anthropologists/sociologists on the villages studied by them, and the fourth one was a full-length monograph – the very first and by an Indian social scientist. In the 1950s, several studies of individual villages were undertaken. Economists and social anthropologists later joined the movement of village studies. The results of these investigations evoked great interest and stressed the necessity for still further study. Lucas in the Punjab initiated intensive studies of particular villages and general agricultural problems. Keatings and Harold Mann in Bombay, Gilbert Slater in Madras, and E.V. As a result, the cultivator of the soil began to attract considerable attention from students of Indian society. Patel, in his book Agricultural Laborers in Modern India and Pakistan, talks about the growth of village studies: With the end of the First World War, the beginnings of an agrarian crisis was accompanied by the entry of peasants into the political arena, as exemplified during the Champaran and Kaira campaigns led by Gandhiji. It felt the need to investigate the actually existing conditions. The growing rural discontent also worried the British Government. Social scientists also became interested in studying rural problems, particularly the deteriorating rural economy. Gandhi organized mass movements to draw attention to the problems of the rural people, and also to involve the peasants in the freedom struggle. Illiteracy, ignorance, and poverty characterized the vast population of rural India. The development of Village India, for Gandhi, was the development of India. It was the villages in which 89 per cent of the population lived. Mahatma Gandhi is often quoted as having said: “Real India lives in its villages.” The fact that in the early decades of the 20th century, India’s urban segment constituted only 11 per cent of the total population gave strength to his argument. Difference between Villages in Ancient India and Villages of Today! What's more, in the EU, this dummy is required in just one of five regulatory tests, and only in the passenger seat. In reality, this dummy is simply a scaled-down male dummy-and women are not scaled-down men. In the last few years, car regulators seem to have belatedly realized that women exist, and have since introduced what they call a “female” car crash test dummy. In the 1950s, when car crash test dummies were first introduced, the dummies were based around the 50th-percentile male, as if he were the 50th-percentile human. In the 1930s, the influential Swiss architect Le Corbusier came up with “the human scale” for architecture, and by "human" he meant a six-foot man with his arm raised. What does the default male body look like? And how are you able to determine if something was designed for a “default male” body? Men (and women, and people of all gender identities) obviously come in different shapes and sizes. You mention the “default male” in the book as being the body type for which so many things are designed, from seat belts to cell phones. And so tech for women remains, for the most part, of the shrink it, pink it and price it up variety, rather than genuinely catering to women’s needs. They are therefore less likely to get funding. As a result, entrepreneurs developing new tech for women need good data because they will not be able to rely on a VC already having personal experience with, for example, how terrible all the current breast pump options are.īut because we lack data on female bodies, such entrepreneurs are less likely to have the information they need to make the case for their ideas. Ninety-three percent of venture capitalists are men, and these teams suffer from the same problem as male-dominated developer teams: they simply aren't aware of certain female needs. Tech is littered with examples of how this plays out, from Apple’s “comprehensive” health app that you could use to track your copper intake but not your period, to step tracker apps that forget women usually don’t have pockets big enough to carry their phones on them at all times. And so the tech that they develop will inevitably be biased towards white middle-class men from America. This is a data gap in its own way: white middle-class men from America simply cannot be aware of the needs of all of humanity. The other issue is how male-dominated tech is. If they had designed their schedule based on sex-disaggregated travel and hospital admission data in the first place, they could have saved a lot of money over the decades. Because it wasn’t men in their cars who were falling over and fracturing their bones: it was women pushing buggies through the snow. So they decided to switch the order around-and found to their surprise that the number of admissions to the emergency room fell dramatically. As a result, the order in which the snow was being cleared (major roads first local roads and sidewalks second) benefitted men. They make lots of short interconnected trips, and are more likely to use public transport. But because women have to combine their paid work with their unpaid care work (women still do 75 percent of the world’s unpaid care work), their travel patterns are more complicated. Men tend to have much simpler travel patterns than women: a twice daily commute in a car. When local officials in the town of Karlskoga in Sweden looked at their snow-clearing schedules, they realized that they had designed them to meet the needs of men. How, exactly, did you notice this showing up in your research? Can you give us an example that really surprised you? In the book, which cites hundreds of studies from around the world, you talk about how women are systematically excluded from data gathering. And not forcing them to be something that they’re not. Allowing women to get older and be happy. MC: You’re so associated with the L’Oréal brand-to be so forward with your natural hair color reflects the best of what the beauty industry is doing now.ĪM: I agree with you. I was never conscious can’t say we have salt-and-pepper hair! I now say I have salt-and-pepper hair, and someone just said to me that’s only been used for men, that expression. I just think this is also a lovely option. I’m not anti-color you have to do what makes you happy. MC: Did letting your hair go gray enhance your confidence?ĪM: I had been wanting to do it, so COVID did me a favor. And being comfortable makes you more confident, too. It makes me uncomfortable trying to pretend that I’m younger. MC: Even of just saying your age out loud.ĪM: Oh, yeah. We have this instilled fear of getting older. We’re so used to, as women, being defined by being ageless. You don’t know how that’s going to be treated in Hollywood. But there’s something else-I think me being comfortable with stepping into my age is beneficial. Marie Claire: You’ve been a working actor for many years, but it seems like there’s an Andie MacDowell renaissance brewing lately-do you feel that?Īndie MacDowell: Work generates work-that’s always been a pattern. We spoke to the likely Emmy nominee and long-time L’Oréal brand ambassador about embracing her grays, teaching her daughters self-care, and hanging with the Euphoria kids. Today, after divorcing Qualley, and a brief marriage to businessman Rhett Hartzog, MacDowell is happily living on her own, back in L.A. Then, in 1999, she relocated to Asheville, N.C., to raise her two daughters, Margaret and Rainey, and son, Justin, with her former model husband Paul Qualley. In 1991, after starring in Steven Soderbergh’s Sex, Lies, and Videotape and just before Groundhog Day and Four Weddings and a Funeral, MacDowell moved to Montana. Even at the height of her fame, MacDowell seemed more grounded than many in her line of work. Audiences have always known her with her iconic chestnut curls, and in Hollywood, where youth is king, it felt like a bold step forward for age positivity.īut perhaps that should be no surprise. A few months before the series premiered, MacDowell debuted her natural gray hair at Cannes. Her character is fiery and heartbreaking, with a brilliant silver mane, showcasing a different side of the actress whose ethereal beauty and Southern lilt made her an ’80s and ’90s movie muse. In it, MacDowell is Paula, the undiagnosed-bipolar mother of domestic-abuse survivor Alex (played by Margaret Qualley, MacDowell’s real-life daughter). Last fall, she gave an unforgettable performance in the Netflix series Maid, based on the memoir by Stephanie Land. 1997! I was in my !” The actress, now 64, is getting the last laugh about that hilariously premature lifetime achievement award (for which, she adds, “I’m not ungrateful!”). I got this award in 1997-for my body of work. She comes back to our Zoom conversation holding an ornate gold statuette: It’s a César, the French equivalent of an Oscar. “I have something funny to show you,” Andie MacDowell says, disappearing out of frame for a minute. By submitting your information you agree to the Terms & Conditions and Privacy Policy and are aged 16 or over. “You have the opportunity to do more complex and deeper work, like the things I had to look for in Paula.There was a problem. “What’s interesting now is that I can play character roles like Paula, which are honestly so much more fun and interesting. “And it’s true after 40, it gets really hard for women - where it gets really interesting for men - and jobs become much harder. “My 30s were so easy - I was turning down work because I wanted to spend time with my children,” she recalled. MacDowell, long after box-office hits like “Sex, Lies and Videotape” (’89) and “Four Weddings and a Funeral” (’94), has survived, thrived even. It’s almost like she was born with this wisdom.” “Her daughter is emotionally balanced and doesn’t have the problems that Paula has. She makes life very difficult for her daughter as well. I mean she’s got her demons and it’s not been easy for her. She doesn’t have those kinds of luxuries in her life. “She’s not on medication because she doesn’t have a doctor. She’s doing the best she can under the circumstances. “We don’t really know what her complete backstory is but I don’t think that it’s good. I used the word ‘crazy’ to describe her the other day and regretted it because I don’t really think it’s the right term for her,” MacDowell said. Community content is available under CC-BY-SA unless otherwise noted. Don't need to change any configuration in the files from the three mods, just start or load a game and the above items will render invisible. Hydrofuge is an item acquired from pestilent (and rather dangerous) Pond Skaters.Breaking a Balsam log will grant the player a Resinless Balsam log and up to three Tree Sap. jar.Mushroom Quest.This mod adds content to the End (biomes, mobs, blocks, etc. Two of the defining features of a Balsam tree are the branches and the bark the branches grow from around 3 blocks up all the way to the top, in an alternating stairway-like pattern, and the bark has the orange of leaking tree sap visible all through it. Install JEI for help search the items, but is optional. Bamboo trees yield bamboo stalks instead of blocks of raw wood.Once defeated, the Brood Mother spawns the egg along with a Tarantula Boss Trophy shirt. Los bambúes de Minecraft también se utilizan como combustible en. The Tarantula Egg is, as of the current version, an aesthetic trophy similar to the Enderdragon Egg, granted by defeating a Tarantula Brood Mother in her nest atop a Giant Baobab Tree. Se puede utilizar para fabricar andamios y palos. Place nine pieces of bamboo into the crafting grid, one in each slot. Bamboo Jungle (Imagen a través de Sportskeeda) Los bambúes de Minecraft se utilizan en varias cosas, que incluyen: Cultivo de granjas de bambú rompiéndolo y plantándolo en el lugar deseado. Install the Erebus, version 1.0.30, with the OpenComputer, version 1.7.4.153 (the last version in CurseForge) and OpenSecurity, version 1.0-35 (the last version in CurseForge). Craft a Block of Bamboo: Open your crafting table.The above items render invisible with OpenSecurity. Now how many valence electrons are allocated to the bonded atom? Well one way to think about it is, draw a circle around thatĪtom in the molecule, and you want to captureĪll of the lone pairs, and you want to capture, Periodic table of elements, free neutral hydrogen So what's the number of valence electrons in a free, neutral atom of hydrogen? Well we've seen this multiple times, you could look at this on the So lets try and make sense of this by applying thisĭefinition of formal charge to the constituents of nitrous acid. Number of lone pair electrons, number of lone pair electrons plus one half of the So if we want to thinkĪbout the valence electrons that are allocated to a bonded atom, these are going to be the And so you're next question is, what does is mean to be allocated? Well, I will break up thisĭefinition a little bit. To subtract the number of valence electrons allocated, allocated to bonded, bonded atom. To calculate the number of valence electrons in free, in free neutral, neutral atom, atom. So the definition of formal charge, and we're going to do thisįor each atom in our molecule, for each atom, we're going To the resonance hybrid based on formal charge. Resonance structures for nitrous acid, but we'll think about which one contributes more These are both legitimate Lewis diagrams. We're going to calculate the formal charge on the various atoms in each of these resonance So before going too deep into that, let's just give ourselves aĭefinition for formal charge, and then as practice, Molecule will contribute most to a resonance hybrid. Which resonance structures, which configurations of a That we can calculate for each of the individualĪtoms in a molecule, and as we'll see in future videos, it'll help us think about The molecule as a whole, it's actually a number Is a tool that we can use as chemists to analyze molecules. The idea of formal charge, and as we will see, it Write the Lewis structure and chemical formula of the compound with a molar mass of about 70 g/mol that contains 19.7% nitrogen and 80.3% fluorine by mass, and determine the formal charge of the atoms in this compound.- In this video, we're going to introduce ourselves to. Write Lewis structures for each of the four compounds and determine the formal charge of the iodine atom in each molecule: Iodine forms a series of fluorides (listed below).Is the actual structure consistent with the formal charges? Draw the structure of hydroxylamine, H 3NO, and assign formal charges look up the structure.Based on formal charge considerations, which of the following would likely be the correct arrangement of atoms in sulfur dioxide: OSO or SOO?.Based on formal charge considerations, which of the following would likely be the correct arrangement of atoms in hypochlorous acid: HOCl or OClH?.Based on formal charge considerations, which of the following would likely be the correct arrangement of atoms in nitrosyl chloride: ClNO or ClON?.Determine the formal charge on each atom in each of the resonance structures: Draw all possible resonance structures for each of the compounds below.Thus, we calculate formal charge as follows: Another way of saying this is that formal charge results when we take the number of valence electrons of a neutral atom, subtract the nonbonding electrons, and then subtract the number of bonds connected to that atom in the Lewis structure. The formal charge of an atom in a molecule is the hypothetical charge the atom would have if we could redistribute the electrons in the bonds evenly between the atoms. We can use the concept of formal charges to help us predict the most appropriate Lewis structure when more than one is reasonable. As we have seen, however, in some cases, there is seemingly more than one valid structure for a molecule. In the previous section, we discussed how to write Lewis structures for molecules and polyatomic ions. Explain the concept of resonance and draw Lewis structures representing resonance forms for a given molecule.Use formal charges to identify the most reasonable Lewis structure for a given molecule.Compute formal charges for atoms in any Lewis structure. Then in the conference finals they would be victorious over the Seattle SuperSonics in a classic seven game series. In the second round they would beat the David Robinson led San Antonio Spurs, 4-2. The first round with the maximum five games as they defeated the Los Angeles Lakers, 3-2. The playoffs would be filled with battles. That offense helped the Suns have the best record in the NBA, 62-20. Barkley led this team to the top ranked offense in the NBA. He was also very efficient, shooting 52% from the floor. Barkley would win the MVP award as he averaged 25.6 points and 12.2 rebounds per game. This was a genius move as Barkley had his best season in his first year with Phoenix. Prior to the 1992-93 season, the Suns traded Jeff Hornacek, Tim Perry, and Andrew Lang to the Philadelphia 76ers in exchange for Charles Barkley. Phoenix would take a 2-0 lead in the series but go on to lose four straight. They would go on to meet the Milwaukee Bucks in the NBA finals. In the western conference finals, the Suns defeated the Los Angeles Clippers in six games. This guy created a toy of the "Suns in 4" fan that went viral Making a Suns’ fan a celebrity in the process. In the second round the Suns would sweep Nikola Jokic and the Denver Nuggets. LeBron James and the Los Angeles Lakers were no match for the Suns in the first round of the playoffs. Booker averaged 25.6 points per game as he shot 48% from the floor. Paul averaged 16.4 points and 8.9 assists per game, while shooting 49% from the floor and 39% from three. This team was led by Chris Paul and Devin Booker. They scored 115.3 points per game while only giving up 109.5 points per game. The 2020-21 Phoenix Suns were a very balanced team as they ranked seventh in points per game and opponent points per game. In the conference finals they would lose in five games to the eventual NBA champion San Antonio Spurs. In the second round they defeated Dirk Nowitzki and the Dallas Mavericks in six games. The Suns started the playoffs of by sweeping the Memphis Grizzlies. The leading scorer on the team was Amar’e Stoudemire who averaged 26 points per game on 55% shooting from the field. This was the first MVP award for Nash, who averaged 15.5 points and 11.5 assists per game. Nash led the Suns to the best record in the NBA, 62-20. The 2004-05 Phoenix Suns were led by Most Valuable Player Steve Nash. The Lakers went on to win the NBA championship. Then ran into Kobe Bryant and the Los Angeles Lakers in the conference finals. In the second round they swept the Tim Duncan led San Antonio Spurs. Phoenix beat the Portland Trailblazers in six games to start the playoffs. He averaged 16.5 points and 11 assists per game, while shooting 50% from the floor, 42% from three, and 93% from the free throw line. Steve Nash at age 35 was still one of the best point guards in the NBA. He was also wildly efficient shooting 55% from the floor. Amar’e Stoudemire played all 82 games during the regular season and averaged 23.1 points per game. The offense carried them to a 54-28 record. They were ranked first in offense, scoring 110 points per game. The 2009-10 Phoenix Suns were another great offensive team in the valley. In the western conference finals, they were swept by Magic Johnson, James Worthy and the Los Angeles Lakers. After that they defeated the Chris Mullin and Mitch Richmond led Golden State Warriors in five games. In the playoffs, the Suns swept the Denver Nuggets in the first round. Phoenix also had great scoring off of the bench as Eddie Johnson averaged 21.5 points per game. Johnson was also very efficient, he shot 50% from the floor. Kevin Johnson had a great all-around season as he averaged 20.4 points and 12.2 assists per game. Tom Chambers was the leading scorer on this squad. The offense carried this team to a 55-27 record. The Suns lead the league in scoring that season. The 1988-89 Phoenix Suns were machine like on offense as they scored at will. Phoenix would lose in the NBA finals to the John Havilek and Dave Cowens led Boston Celtics. After that they would go beat the 59-23 Golden State Warriors in the western finals. They found their groove in the playoffs as they defeated the 43-39 Seattle SuperSonics in six games in the western conference semi-finals. Westphal averaged 20.5 points per game and Adams scored 19.5 points per game. The leading scorers on this team were Paul Westphal and Alvan Adams. Despite their offensive and defensive ratings, the Suns ended the regular season with a record of 42-40. The 1975-76 Phoenix Suns were ranked eighth in the NBA in points per game scored and opponent points per game. Who are the top five teams in Suns’ history? Honorable Mention: 1975-76 They have not won a championship in team history but have had lots of great teams. The Phoenix Suns have a rich history as a franchise. He sees himself as this liberator, he's fully deluded in this self-assessment. I think Sam sees himself as a Travis Bickle. I thought, well, I love The Goonies, and I love David Lynch, and I love Los Angeles.Īlso I loved it because it was quite skewering of Hollywood and a kind of patriarchal abusive system. And I read it and I just thought, this is totally marvellous and totally unique, and as I'm sure you hear all the time and that you feel as a film journalist, it's very, very rare that you get a unique piece of cinema in this day and age. But now he's in a David Lynch film that's set in a Los Angeles that's like the antithesis of the La La Land Los Angeles. It was 160-odd pages, which is about 30 or 40 pages more than the usual script, and it was like- I like describing, it's as if Sean Astin's character, Mikey, from The Goonies is far too old to still be going on adventures, but he still wants to. I got sent the script for Under The Silver Lake and it was as mind-bending in word, as it was, in deed, as it were. I was just so impressed with him as a filmmaker. And then of course It Follows was just this kind of genre reinvention, in the horror genre. I'd seen It Follows and I loved it, and I had seen The Myth Of The American Sleepover and I thought it was so unique and specific in its vision. What was David Robert Mitchell's pitch to you?ĪNDREW GARFIELD: He didn't really have to pitch it to me. Sam is a pop culture obsessed guy on this weird journey into. Listen to the interview here, and read the full conversation below.ĮMPIRE: Under The Silver Lake is an absolute trip of a film. He stopped by the Empire Podcast to talk all things silvery and lake-like, as well as divulging his personal pop culture obsessions and reflecting on his hugely impressive filmography. And in his latest role, Andrew Garfield is Sam, a pop culture obsessed slacker on a down-the-rabbit-hole neo-noir quest in David Robert Mitchell’s Under The Silver Lake. He’s been a Portuguese missionary in 17th century Japan. Vlasuk, G.P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. Directed deletion of a yeast transfer RNA intervening sequence. Wallace, R.B., Johnson, P.F., Tanaka, S., Schold, M., Itakura, K., and Abelson, J. In: Experimental Manipulation of Gene Expression, Inouye, M. Site specific mutagenesis using synthetic oligodeoxyribonucleotides as mutagens, p. Constructed mutants using synthetic oligodeoxyribonucleotides as site-specific mutagens, p. The present method can be applied to any gene cloned in a plasmid vector and is more convenient and simpler than the method using an M13 phage vector, since the mutated gene can be expressed directly from the plasmid vector used for the mutagenesis. In the case of a single base substitution, the yield of the desired mutant was as high as 13%. The mixture of renatured DNA was treated with the Klenow fragment of DNA polymerase I plus deoxynucleotide triphosphates in the presence of T4 ligase, and ampicillin-resistant transformants were isolated. Both fragments were mixed with a synthetic oligonucleotide (the mutagen) and the mixture was denatured at 100☌ for 3 min, followed by gradual cooling to reanneal. Another sample was digested by Xbal which cuts at a unique site in the pIN-II vector, and EcoRI to remove the portion of the lpp gene to be mutagenized. This plasmid (5.6-kb) was digested by PstI followed by exonuclease (Klenow fragment of DNA polymerase I) to remove a portion of the gene for β-lactamase. Conditions which gave a high yield of oligonucleotide-directed site specific mutagenesis were obtained with use of the lpp gene of Escherichia coli cloned in the versatile expression plasmid vector, pIN-II. |
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