![]() Simply add 1µl of the second restriction enzyme and adjust the amount of water used to maintain a 20µl reaction. If both restriction enzymes work in the same restriction enzyme buffer, the reaction is straightforward. Preparing an insert for transfer from one vector to another usually requires digestion with two different REs. Add 4µl of 6X Blue/Orange Loading Dye and analyze digested DNA by gel electrophoresis. Incubate at the appropriate temperature and the appropriate time for the enzyme. Mix by pipetting and collect the contents at the bottom of the tube. For a digestion with a single RE the reaction is very simple: Restriction digestion is one of the most common reactions performed in molecular biology. Many restriction enzymes will not cut DNA that is methylated on one or both strands of the recognition site, while some require substrate methylation. Type II REs cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. Restriction endonucleases (RE), also referred to as restriction enzymes, are proteins that recognize short, specific (often palindromic) DNA sequences. Many strategies have been employed to do partial digests including decreasing reaction temperature, using a non-optimal buffer, and decreasing units of enzyme. You can manipulate the restriction digest conditions such that you will digest only a subset of sites. ![]() Under normal restriction digest conditions, the enzyme is in excess so that all recognition sites in the plasmid can be cleaved. The presence of the same restriction enzyme recognition site in the insert and the multiple cloning region does not necessarily preclude use of that restriction site in a subcloning strategy. Subcloning Strategy: Common Restriction Sites with Partial Digests Controlling Cut Frequency in Restriction Digestion Partial Restriction Digestion can be performed. ![]() Having a restriction site in both the multiple cloning region and the insert does not exclude the use of this site for subcloning. ![]() These nicks will be repaired in the bacteria upon transformation. The vector has been dephosphorylated so the second bond will not be formed in vitro (indicated by the OH). The T4 DNA Ligase will join the DNA by reforming the bond between the 5´-PO 4 coming from the insert and the 3´-OH of the vector. ![]()
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![]() ![]() Arrange the chip parts by swapping them until all are aligned correctly.Click on it to open it, triggering a puzzle. Take a closer look at the small square panel beneath the door handle on the left.Click on the Chestnut Lodge Mental Hospital brochure to open it and note the number “962” inside (this will be added to your Journal), then click on the MOTH to add it to your inventory. Examine the magazine rack on the right.Attempt to open it and note that the hinges are rusted. Examine the door in the far left corner.Click on the PAPERCLIP to add it to your inventory. Examine the first table for a closer view.Input the code “6281” (the number on the license plate) to open the door. Take a closer look at the barred door ahead.He needs a glass of water (we’ll come back to this later). Use the ASYLUM KEY on the lock to unlock the door, then proceed inside. Upon completing the area, the ASYLUM KEY will be added to your inventory.This will trigger a hidden object area on the right. Attempt to enter the Asylum, only to have the door slam shut.Note the trunk of the car on the right, and the flashlight on the ground on the path towards the asylum, then continue forward.Upon completing the area, the EMPTY GLASS will be added to your inventory.Click on the hidden object area over the fountain.Once he’s gone, examine the license plate on the ground between the two parking spaces. Use the SLEDGEHAMMER to break through the wall, then continue through the hole to the next area. Examine the brick wall on the left again.Upon completing the area, the SLEDGEHAMMER will be added to your inventory.Objects listed in blue are shown below in blue. Find the objects listed at the bottom of the screen.Exit the close-up to see a hidden object area appear on the right. You’ll need something to break through it. Click on the Morphing Object to collect it.you may get the MAGNET before the EMPTY GLASS, which will result in you being unable to progress further in the game without starting over. In some rare cases, it is possible for items you will need to acquire to disappear entirely/become unobtainable if you do not encounter them in the right order, i.e. ![]()
![]() ![]() The puppy wants to play and make the girls happy. And Sheffield knows that (some) girls like excited puppies. And Sheffield loves music all across the board, like an excited puppy, whether Debbie Gibson or Lita Ford or Big Daddy Kane. Never in a million years would I have admitted, at age 18, to liking Poison. Sheffield likes to portray himself as a wuss with a weakness for shiny pop but he's way more brave than me. The book also reads like the memoir of a polite, Catholic indie-rock fan who fears/adores his three sisters, sells ice cream from a truck, and really, really wants a girlfriend. Rob Sheffield's Talking With Girls About Duran Duran is less about Duran Duran and more about the ways boys in the 80s (and any era, really) accessed music to help them articulate what they can't quite say to girls or each other. Needless to say, I guess I'm not the audience. I just kept being reminded of every douchey guy I've ever met that has talked down to me about music. I enjoyed his previous book- Love Is a Mixtape-but this one's a big mixed bag that just put me in a solemn mood by reminding me that rock journalism is still a big f-ing boys club. I had to give up about halfway through, but not before skipping to the Replacements chapter out of curiosity because I love that band. I enjoyed the funny bits interspersed throughout, particularly where he's talking about his family life and the times where he made it more personal rather than just merely a longwinded pop reference wankfest. Believe me, I'm as music-obsessed as they come, but Sheffield's constant need for dropping pop culture references and lyrics into his writing gets a little tiring in this instance, just like with Chuck Klosterman and Nick Hornby (although they do it a little better.) ![]() His ego takes control of the book, spitting out some witty one-liners every now and then that my younger self would perhaps be more fond of. Sadly, the stereotyped gender statements continue, which just end up getting on my nerves. So, with that having rubbed me the wrong way, I cynically read on. (They love "Stand by Me" but they don't care that it's really called "Train in Vain" instead of "Stand by Me.")" It's a sad attempt at humor through perpetuated gender stereotypes and it only serves to anger any women who LOVE the Clash and have tons of music knowledge. But it's tougher to talk to women about the Clash. "This is just the way we dissect the things we love. I should've known what I was getting into, but I found the intro to be extremely patronizing. These songs-and Sheffield's writing-will remind readers of that first kiss, that first car, and the moments that shaped their lives. Sheffield's coming-of-age story is one that we all know, with a playlist that any child of the eighties or anyone who just loves music will sing along with. Girls, every last one of whom seems to be madly in love with the bassist of Duran Duran. These were the years of MTV and John Hughes movies the era of big dreams and bigger shoulder pads and, like any all-American boy, this one was searching for true love and maybe a cooler haircut. When he turned 13 in 1980, Rob Sheffield had a lot to learn about women, love, music and himself, and in Talking to Girls About Duran Duran we get a glimpse into his transformation from pasty, geeky "hermit boy" into a young man with his first girlfriend, his first apartment, and a sense of the world. Now, in Talking to Girls About Duran Duran, Sheffield shares the soundtrack to his eighties adolescence. ![]() "No rock critic-living or dead, American or otherwise-has ever written about pop music with the evocative, hyperpoetic perfectitude of Rob Sheffield." So said Chuck Klosterman about Love is a Mix Tape, Sheffield's paean to a lost love via its soundtrack. The author of the national bestseller Love is a Mix Tape returns, with a different-but equally personal and equally universal- spin on music as memory. ![]() 4/30/2023 0 Comments Dirt 5 trophies ps5![]() ![]() You can choose different options than the ones highlighted below and still have everybody survive the night, as there are a lot of different decisions that only add slight variations to the story. You only need Laura, Jacob, Abigail, Dylan, Ryan, Emma, Nick, Kaitlyn, and Max to survive in order to get the 'Rough Night' Trophy, but if you want to ensure nobody dies in The Quarry except for a few unavoidable deaths, you're going to have to do the QTE's yourself. If you turn on 'Simple QTE's' from the options menu, this won't be a problem - however, succeeding every QTE closes some available paths, and can lead to the death of some secondary characters. It's important to note that even if you make all of the correct dialogue choices, and follow the safest paths, you can still die from a mistimed QTE (Quick time event). Watch on YouTube The Quarry how to save everyone explained ![]()
4/30/2023 0 Comments Desmume pokemon black![]() ![]() The lowered levels are taken into account for prize money (the player will receive less), but not experience (in the example, the player's Pokémon will still receive experience as if they had defeated a level 59 Haxorus). However, their Pokémon's stats are still calculated using the levels from Normal Mode (for example, Champion Iris's Haxorus will appear to be level 55 but still have the stats of a level 59 Haxorus). In Easy Mode, opponents use less competent artificial intelligence and the level of opposing Trainers' Pokémon are lower than usual. Therefore, the only way to play the main storyline with a difficulty other than Normal Mode is to transfer another player's key from a separate DS/3DS system. This awards the player with the Easy Key (Japanese: アシストキー Assist Key) in White 2 or the Challenge Key (Japanese: チャレンジキー Challenge Key) in Black 2.īecause keys are tied to the save file, starting a new game or deleting the save file will also remove any unlocked keys. Only Normal Mode is unlocked by default Easy Mode and Challenge Mode are exclusive to White 2 and Black 2 respectively, and are unlocked by defeating the Champion. There are three different difficulties: Easy Mode (Japanese: アシストモード Assist Mode), Normal Mode (Japanese: ノーマルモード Normal Mode), and Challenge Mode (Japanese: チャレンジモード Challenge Mode). Pokémon Brilliant Diamond and Shining Pearl. ![]() 4/30/2023 0 Comments Jim fink options trading![]() ![]() Regulators should instead be focusing on how to facilitate the effectiveness of that process. These innovations, through their ability to facilitate direct market participation by retail investors have the potential to democratize the capital markets and increase the connections between ordinary citizens and U.S. ![]() Regulators should not be clamping down on the conditions such as commission-free trading, gamified trading platforms, and the expanded use of social media, that have enabled a generation of new investors to participate in the capital markets. More importantly, the debate overlooks potential benefits from retail investing both to investors themselves and the capital markets. It argues that, although retail investing is likely to impact the capital markets, claims about the harms from increased retail investing are overstated. This Article challenges calls for broad-based regulatory reform. Second, they argue that the stock market, in term, needs protection from retail investors. First, critics argue that retail investors need greater protection from the risks of investing in the stock market. Some commentators have greeted retail investing, which has been fueled by app-based brokerage accounts and social media, with alarm and called for regulatory reform. ![]() The GameStop trading frenzy in January 2021 was perhaps the highest profile example of the reemergence of capital market participation by retail investors, a marked shift from the growing domination of those markets by large institutional investors. ![]() 4/30/2023 0 Comments Markdown code snippet![]() ![]() You’ll need to check whether or not the lightweight markup language your application is using supports the extended syntax elements you want to use. Not all Markdown applications support extended syntax elements. These elements can be enabled by using a lightweight markup language that builds upon the basic Markdown syntax, or by adding an extension to a compatible Markdown processor. Several individuals and organizations took it upon themselves to extend the basic syntax by adding additional elements like tables, code blocks, syntax highlighting, URL auto-linking, and footnotes. You can buy me a coffee if you would like to support me to make more great open-source software.The basic syntax outlined in the original Markdown design document added many of the elements needed on a day-to-day basis, but it wasn’t enough for some people. ![]() If you are happy with the extension, please star the repo, and leave a review to help others find it. You can read my article, VS Code keyboard-fu: custom keyboard shortcuts that will boost your productivity. Want to learn more about keyboard shortcuts? If you have a suggestion or find a bug, please file an issue.
4/30/2023 0 Comments Synology drive compatibility list![]() ![]() ![]() To use a Z-Wave USB stick for Z-Wave control, the HA Docker container needs extra configuration to access to the USB stick. Edit Source IP if you like or leave it at default “All”. Create a new rule and select “Custom” for Ports and add 8123. Click “Edit Rules” besides the Firewall Profile dropdown box. This can be found in “Control Panel -> Security” and then the Firewall tab. If you are using the built-in firewall, you must also add the port 8123 to allowed list. Your Home Assistant within Docker should now run and will serve the web interface from port 8123 on your Docker host (this will be your Synology NAS IP address - for example ).Ensure “Run this container after the wizard is finished” is checked and click Done.This configures where Home Assistant will store configs and logs. Then edit the “mount path” to be “/config”. in “docker” shared folder, add new folder named “homeassistant” and then within that new folder add another new folder “config”), then click Select. Within “Volume Settings” click on “Add Folder” and choose either an existing folder or add a new folder (e.g.To ensure that Home Assistant displays the correct timezone go to the “Environment” tab and click the plus sign then add variable = TZ & value = Europe/London choosing your correct timezone. Choose a container-name you want (e.g., “homeassistant”).Within “Network” select “Use same network as Docker Host” and click Next.Move to the “Image”-section of the Docker-app.Wait for some time until your NAS has pulled the image.Find “homeassistant/home-assistant” within registry and click on “Download”. ![]() Launch Docker-app and move to “Registry”-section.Install “Docker” package on your Synology NAS.For details about the package (including compatibility-information, if your NAS is supported), see Onboarding Install Home Assistant Container Synology NASĪs Synology within DSM now supports Docker (with a neat UI), you can simply install Home Assistant using Docker without the need for command-line. ![]() 4/29/2023 0 Comments Free office chat application![]() The program has its own server where a user control everything happening in the chat. Do not require too many system resources. ![]() ProprietaryGroup Chat AppVideo Calling AppInstant MessengerMacWindowsLinuxAndroidiPhoneSelf-Hosted45 alternatives to M圜hat M圜hat is an instant messenger that works in LAN and over the Internet.Suggest and vote on featuresFeem FeaturesIntegrated Chat 2M圜hatIs this a good alternative? Yes NoCopy a direct link to this comment to your clipboard Freemium ProprietaryMacWindowsLinuxAndroidiPhoneAndroid TabletWindows PhoneiPad137 alternatives to Feem Share files offline.2018 13FeemIs this a good alternative? Yes NoCopy a direct link to this comment to your clipboard Freemium Suggest and vote on featuresLAN Messenger Features PortableLAN Messenger can be used from a USB stick or similar with no prior installation.License plate detectionIntegrated File SharingTeam MessagingDiscontinued Sadly, no more releases since Dec. A variety of handy features are supported including notifications, personal and group messaging with encryption, file transfer and message logging. Open SourceInstant 23 alternatives to LAN Messenger LAN Messenger is a p2p chat application for intranet communication and does not require a server. ![]()
4/29/2023 0 Comments Itunes remote buttonm![]() ![]() Maybe you could post screenshots, I believe your solution lies close to the one I found. Maybe you could post screenshots, I believe your solution lies close to the one I found. Started up iTunes on MBP and could acces the iTunes Library located on the iMac, reinstalled Remote. I would urge you to give it another go, I see your frustration with it, but hey, I solved mine through perseverance! I now am able to select and control either Library. Customize button mapping and menu layout to suit your needs. ![]() Clicked that, filled in the four digits, and hey presto, both my iMac and MBP appeared on the iPhone app, as well as the AppleTV! Control your Mac with Siri Remote, iPhone, Apple Remote, iPad, Apple TV, Harmony Remotes. This may sound a bit confusing, but once you get started. Started up iTunes on MBP and could acces the iTunes Library located on the iMac, reinstalled Remote.app on iPhone and proceeded to "add an iTunes Library" through the app's Settings, the four-digit box appeared, and on the MBP's iTunes, the Remote icon button appeared right beneath the play button, where usually the iPhone icon sits when it is connected through USB. When you click a button in the iTunes window on the remote Mac, you’re actually clicking that button on the main (server) Mac. (All devices must be on the same wi-fi network and using the same AppleID for Remote to work, sometimes after an update, or after restarting, they will log on another previously used network if you have more that one. Provides estimates for modules under development and own work. Review relevant requirements, standards, and other documentation to clarify and understand software engineering requirements. ![]() When I got home, I decided to give it another try.Ĭhecked the Wi-Fi network iPhone was on, and it seems it had inadvertedly logged on to my "other" wi-fi network. Develop, implement, and support technical assignments related to Information Model Management. On that note, did you check if your iPhone is on EXACTLY the same Wi-Fi and AppleID? There are two AirportExpress, an AppleTV and an Airport TC taking care of the Wi-Fi (music) distribution around the house, sometimes they play up, but generally it works fine, specially when I can control everything from my iPhone with the RemoteApp. I have never used iTunesMatch or AppleMusic, as everything worked fine for me. In the Music app on your Mac, click the Remote button under Devices in the sidebar, then type the 4-digit code. Just connect Android device and media player to the same Wi-Fi network and you will be able to control it. Open iTunes Remote and choose Set Up Home Sharing. This application allows you to control Apple TV. Hi, I have been using iTunes from the beginning also, and frankly, this is the worst it's been imo. Connect your iPhone or iPad to the same Wi-Fi network where iTunes is connected. ![]() |
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