The length of the homologous ends depends on the number of fragments being assembled and their length (see below). PCR primers need to be designed to amplify your fragment of interest and include 20 or more base pairs of homology to the vector or adjacent fragments in your cloned product. ► Learn to simulate Gibson Assembly in SnapGene Insert Preparation You have the option to prepare your vector by linearizing your plasmid with restriction enzymes or by using Reverse PCR. Insert fragments are always prepared by PCR. To perform Gibson assembly, you will need to prepare one or more inserts and your vector. This article refers to all these reactions generically as Gibson Assembly. Second-generation assembly reactions such as HiFi Assembly and Gibson Assembly Ultra improve on these limitations. The classic Gibson Assembly reaction can result in errors at fragment junctions and does not work with single-stranded fragments or short fragments. The reaction is designed to occur at 50☌, with all enzymes selected for their stability and activity at this temperature. T5 exonuclease chews back the 5’ end of double-stranded DNA to expose engineered overlaps. Polymerase begins filling in at the overhangs preventing excessive enzyme chewing. When stable compatible overhangs anneal, ligase completes the fusion of the DNA fragments. Once ligated, the DNA is protected from the exonuclease. These enzymes work together to fuse overlapping DNA fragments.
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Alternatively, use the excess dough to make designs on the pie.Lay the strips across the top of the pie to make a lattice design. Instead of just covering the pie with dough, why not use a nice lattice-crust design? Just roll your pie dough out on a floured surface and slice it into strips.Thaw: To thaw, put your pie in the fridge the night before.Freeze: To freeze, wrap in plastic wrap and place in a freezer bag.Store: Once the pie is cooled to room temperature, store leftovers in a covered pie pan or another airtight container.Skip the crust and just make the filling part.Our instant pot pineapple chicken is a delicious entree for this pie.You could also toss a handful of chopped pecans or walnuts on top.This scrumptious pineapple pie is the perfect dessert for slow cooker pineapple pork loin.Sprinkle chocolate syrup and chocolate chips on top for chocolate lovers.Check out our delicious recipe for this luscious dessert. Use leftovers to make pineapple crisp.Try my recipe for homemade whipped cream. Top with whipped cream, Cool Whip, or whipped topping for a creamy pineapple pie.Try one of our awesome serving ideas or one of your own. You can make it any time and have this delicious dessert on the table in less than 45 minutes. When you want something sweet and delicious but also a little bit healthy, this pineapple pie is perfect. Creamy pineapple pie: Add a package of vanilla pudding mix instead of cream cheese for an extra creamy dessert.Pineapple extract: For extra pineapple flavor, use pineapple extract instead of (or in addition to) coconut extract.Just replace the pineapple with what you like such as apples, pears, or peaches. Other fruit pies: You can make any kind of fruit pie you want with this recipe.Frozen pineapple: Alternatively, you can use frozen pineapple chunks instead.Fresh pineapple: You could also use fresh pineapple if you have the time and energy to cut it up.Cinnamon stick: For a unique taste, add a cinnamon stick to the mixture.You can use store-bought or your own graham cracker crust. Graham cracker crust: Use graham cracker pie crust instead of regular for a crunchier pie.Lemon: Use lemon juice and lemon zest instead of lime for a slightly different flavor.Cut and serve: Once it is completely chilled and set, it’s time to eat!.Chill the pie: Next, pour the pie filling into a no-bake crust and let it chill in the fridge covered with plastic wrap for at least two hours or overnight.Cream cheese: Beat the cream cheese until creamy, and once the pineapple mixture has cooled, beat together to combine.Bring to a boil over medium heat and then simmer on low for about 10 minutes until the mixture is thick and the liquid is mostly gone. Make the filling: First, combine all of the ingredients except the cream cheese into a medium saucepan.Cool and serve: Last, let creamy pineapple pie cool for 15 to 20 minutes before slicing and serving.Bake the pie: Finally, make sure your oven rack is in the center when placing the pie in the preheated oven and bake for 35 minutes or until the top crust is golden brown.Cover the pie: Next, if you are using a pie shell with a top crust, lay the top over the pineapple pie filling and crimp the edges before cutting a few slices in the top for steam vents.Mix the pineapple filling: Meanwhile, stir all the ingredients for the pie filling in a large bowl and then smooth it into the prepared crust.Preheat: First, preheat your oven to 350 degrees F. Brown sugar: We used light brown sugar.Canned crushed pineapple: Any kind of canned pineapple will do.We have the exact measurements and instructions below on our recipe card. These are the basic ingredients for baking this pineapple pie recipe. Luffy heads to the church, where he finds Gordon being confronted by some Marines over the whereabouts of the Straw Hats. Gordon says that Chopper was last at the church in town, and heads over there himself. Upon arriving at Sogyoku Town, he is confronted by a man named Gordon however, Jeanne then races in to inform Gordon that Luffy is Chopper's crewmate. She guides Luffy down a path going directly to the town, and Luffy is forced to fight and defeat some Marine soldiers on the way the soldiers all carry Seastone bullets which, unlike normal bullets, can harm him. He says he is looking for his crewmates, and Jeanne reveals that Chopper is at the nearby Sogyoku Town caring for injured people there. He asks Luffy if he is coming after the stones, but Luffy has no idea what those are. Jeanne reveals that she rescued Luffy, and Doni expresses apprehension at what she did, believing Luffy might be a violent pirate. Luffy wakes up on the beach, where he meets a young woman named Jeanne and a little boy named Doni. However, Isaac then sends his gauntlet flying down to strike him from above, sending Luffy plummeting straight into the ocean. As Luffy falls hundreds of meters through the air, he balloons himself to control his descent and ensure he lands on land. This distracts Isaac just enough to allow Luffy to free himself, and Luffy immediately proceeds to jump down toward Jail Island. However, right as the warden has pinned the pirate to the ground, a Marine comes out and informs him that a certain woman wishes to speak with him. Isaac manages to overpower Luffy with his mechanical enhancements, including a gauntlet over his arm that can fly and be controlled remotely as well as rocket jets allowing him to fly. Zoro and Sanji then leap off the Sky Prison and fall down to Jail Island, but Luffy stays behind to confront Isaac. Zoro and Sanji, who are in disguise as Marine guards on the pavilion, proceed to drop their charade and take down the Marines guarding Luffy as Robin sprouts an arm to unlock Luffy's handcuffs. As Nami hurriedly informs the others, Usopp, Franky, and Trafalgar Law are keeping watch over the Thousand Sunny and are suddenly forced to defend it from an assault by robot warriors. However, upon opening it they are met with a shock, as nothing is in the safe and their mission was a trap. As Robin and Chopper ambush the guardroom to seize the key to Luffy's Seastone handcuffs, Nami and Brook infiltrate the prison and open the safe they believe the treasure to be in. However, Luffy had gotten captured intentionally as part of the plot by the Straw Hat Pirates to find a treasure they had heard about in the Sky Prison. The guards take him to an outdoor pavilion, where the warden Isaac greets him and welcomes him to Jail Island. The story begins with Luffy being led through a corridor by Marine guards in the "Sky Prison" high up in the air. Story World Seeker Prologue: "To Sogyoku Town!" Techniques from the anime like Gum Gum Rocket which allows Luffy to easily traverse the world and Gum Gum Bazooka are also featured in the game. Among other elements, the combat features stealth elements and Luffy can use the Kenbunshoku Haki to see enemies from behind walls. It is set in Prison Island, and the player can experience and wage battles in an open world environment which can be freely explored. World Seeker puts players in the role of Luffy from a third-person perspective. 4chan Logo Meme board, clover transparent background PNG clipart size: 900x900px filesize: 372.66KB.wechat logo, iPhone iMessage Messages Logo Computer Icons, message transparent background PNG clipart size: 512x512px filesize: 24.88KB.pen nib, Pen Tool Computer Icons, fountain pen transparent background PNG clipart size: 1024x1024px filesize: 12.21KB.Telegram Logo, Sticker, Telegram Bot Api, Instant Messaging, Line, Blue, Aqua, Azure transparent background PNG clipart size: 512x512px filesize: 53.97KB.
During a self-exam, you may feel a thick area of irregularly shaped tissue with a lumpy or ridge-like surface, or you may encounter a bead-like texture to your breast tissue. How can you tell? Symptoms include a dense, irregular, and bumpy consistency of the breast tissue. If your breasts feel lumpy, and you have intermittent breast discomfort, such as tenderness, swelling, and pain, you may have fibrocystic breast disease. Dorling Kindersley / Getty Images/Dorling KindersleyĪlthough the term fibrocystic breast disease may sound ominous, it actually describes a benign condition of the breasts that more than 60 percent of all women experience. You can also find us on Facebook, Twitter and Google+.Symptoms of fibrocystic breast disease include a dense, irregular, and bumpy consistency of the breast tissue. If you liked this video, then please subscribe to our YouTube Channel to get updates of other useful health video tutorials. 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Here's how social media reacted to Miller's claim. Coach Mike Budenholzer said the X-ray on Antetokounmpo was "clear" and the team will monitor his back heading into Game 2.īut is losing Herro more significant to the Heat's chances in a game or series than it would be for the Bucks to lose Antetokounmpo, a two-time MVP, the Bucks' all-time leading scorer and a player who alters the course of a contest with the attention defenses give him? The Bucks were 11-8 when Antetokounmpo didn't play this season, something Miller cited in his take, but two of those losses were to the Heat this season in Miami, where the series will shift for Games 3 and 4. Well, Miami received contributions from up and down its lineup, most notably from four-time All-NBA player Jimmy Butler (35 points), 2023 All-Star Bam Adebayo (22 points) as well the ageless Kevin Love, who poured in 18 points with one huge 3-pointer after another.Īntetokounmpo's status is less clear than Herro's. "Where does (coach Erik) Spoelstra go now because he’s done for the rest of the series, Herro? We assume Giannis is coming back at some point in this series." "It’s a bigger loss not having Herro for Miami, his ability to handle the basketball, shoot the basketball, pretty good at the defensive end," said Miller, who was calling the game for TNT with Kevin Harlan. Herro, the third-leading scorer for the Heat during the regular season at 20.1 points per game, helped the Heat get off to a fast start against the Bucks in Game 1 with his 12 points on 5 of 9 shooting before his injury. If you ask Reggie Miller, it was Herro's injury. So, which one was more significant to their teams' chances at winning? Miami's Tyler Herro, meanwhile, suffered a broken right hand late in the second quarter while going for a loose ball and will miss the rest of the series. Milwaukee's Giannis Antetokounmpo suffered a back contusion and left the game for good early in the second quarter and the Bucks never could recover in a 130-117 loss. Sunday's game between the Milwaukee Bucks and Miami Heat in the first round of the 2023 NBA playoffs featured significant injuries for both teams and each could be series altering. Watch Video: Bucks monitoring Giannis as they prepare for Game 2 versus Heat Continuation fom previous ArtifIce thread. Continuation of the previous ArtifIce thread, fleshing out game mechanics some more and adding a little fluff.Ĭontinuation of the previous ArtifIce threadĪrtifice 3 AI with a vengeance. In 1934, an aircraft manufacturer approached the George Tucker Eyelet Company with a challenge: Could it design a rivet that could be set from one side, particularly if there was no access to the other side of the material? 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A rivet that’s too narrow for its hole can leave a gap, which can increase shear stress or cause installation problems also, the blind head could expand in the space between the two workpieces. If a rivet is too wide for its hole, it will be harder to place and result in a poor fit. Carefully measure hole sizes, and never force a pop rivet into anyplace that doesn’t match its diameter. Body Lengthīody length is the total length of the pop rivet (not including the head). It differs from grip range (which will be explained next), but is an important measurement when considering clearance on the blind side of the material being fastened-a rivet that’s too long may not properly fit or could negatively impact whatever resides behind the hole. Grip range is the thickness of material that a rivet can effectively fasten. If, for example, you are attaching two quarter-inch pieces of metal together, you will require a pop rivet with a minimum grip of at least a half-inch. When considering grip range, best practice dictates a rivet that isn’t directly on the minimum or maximum points, but rather, comfortably in-between. This ensures shear and tensile strength and improves the longevity of the setting tool. Head Sizeįlat head pop rivets are flush with the material being fastened. The heads of dome rivets reside above the material, and large flange rivets take the concept further by covering more area and providing extra fastening capabilities. Paying attention to head size helps with determining integrity, protrusion, and aesthetic value. The Right Rivet for the Jobĭetermining the correct size of pop rivets for the job is a step that cannot be overlooked, but equally important is picking the right rivet style for your application. Some common types include:Ĭlosed end rivets : These pop rivets come in two pieces and offer great versatility, strong vibration resistance, and clean installations. Open end rivets : Also called nail rivets, open-end rivets differ from their closed-end counterparts by not offering a prevention seal against liquid or vapor escape. Micro rivets : With a diameter of 2 millimeters or smaller, micro rivets are perfect for small applications such as printed circuit boards and other electronic applications. Blind Standoffs for Stainless Steel Sheets.Large Head High-Torque Self Clinching Studs.Self-Clinching Studs for Stainless Material.Miniature Self-Locking Self-Clinching Nuts.Hardened Nuts for Stainless Steel Sheets.Pop rivets come in many styles, including closed-end, open-end, micro, and multi-grip.Hole size, body length, grip range, and head size must be properly measured to determine the proper size for pop rivets.Invented in 1934, pop rivets give users to ability to place a rivet from one side of the material.Multi-grip rivets : This option provides a wider grip range than standard pop rivets and can accommodate oversized and irregular-sized holes.Ĭorrectly assessing size and type of pop rivet, along with the material the rivet is made of, maximizes the success of your application and ensures the rivet can endure the rigors of long-term use. |
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