Successful modification of the bacterial artificial chromosome (BAC) after two-step BAC engineering is confirmed in two separate polymerase chain reactions (PCRs). The first reaction (5' co-integrate PCR) uses a forward 5' co-integrate primer (a sequence located upstream of the 5' end of the A-box) and a reverse 3' primer on the vector (175PA+50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3' co-integrate PCR) uses a forward 5' primer on the recA gene (RecA1300S) and a reverse 3' co-integrate primer (a sequence located downstream from the 3' end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including sacB and recA genes become UV light sensitive.

Bacterial artificial chromosome (BAC) clones are rendered electrocompetent and transformed with the recombinant shuttle vector, pLD53SCAB/AB-box. Cointegrates are selected by growth on chloramphenicol and ampicillin to ensure recombination of the shuttle vector into the BAC.

Plasmid DNA is prepared from the recombinant shuttle vector pLD53.SCAB/A-B created by cloning of the A and B homology arms for two-step bacterial artificial chromosome (BAC) engineering. To confirm that the A-box and B-box arms have been successfully incorporated into pLD53.SCAB, the pattern of enzyme digestion of the modified plasmid is compared with that of the unmodified pLD53.SCAB. Once the shuttle vector is shown to carry the proper sequences, it is ready for transfer into the BAC host.

This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector.

The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector.

In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The shuttle vector pLD53.SCAB (or pLD53.SCAEB) carries the recA gene and the R6K origin, which requires the protein to replicate. PIR2 cells, expressing , are typically used for the amplification of the vector and maintain about 15 copies/cell of the donor vector, which is relatively stable in this host.

BIS Bulletin No 7, April 2020. Past epidemics had long-lasting effects on economies through illness and the loss of lives, while Covid-19 is marked by widespread containment measures and relatively lower fatalities among young people. The short-term costs of Covid-19 will probably dwarf those of past epidemics, due to the unprecedented and synchronised global sudden stop in economic activity induced by containment measures. The current estimated impact on global GDP growth for 2020 is around -4%, with substantial downside risks if containment policies are prolonged. Output losses are larger for major economies.

This paper studies the relationship between bank geographic complexity and risk. We use a unique dataset of 96 bank holding companies around the world to measure the geographic dispersion of their affiliates. We study how this dispersion interacts with economic and regulatory conditions to affect the riskiness of the bank.

BIS Bulletin No 8, April 2020. Information on local labour markets and Google searches can be used to construct a measure of the vulnerability of employment in different regions of the United States to the Covid-19 shock. Regional exposure to Covid-19 varies significantly, ranging from a low of 2% to a high of 98% of total local employment. We test for the usefulness of the Covid-19 exposure measure by showing that areas with higher exposure report more Google search queries related to the pandemic and unemployment benefits.

Our ongoing celebration of the Library of Congress National Book Festival continues with Poets Laureate Tracy K. Smith and Robert Hass discussing the making of poetry, the position of Poet Laureate and their new books, "American Journal: Fifty Poems for Our Time" (edited by Smith) and "A Little Book on Form: An Exploration into the Formal Imagination of Poetry" (Hass), on the Poetry & Prose stage at the 2018 Festival.

The graphic designer is receiving a Lifetime Achievement Award from Cooper Hewitt for her recognizable computer icons, typefaces and graphics

Press release "Governors and Heads of Supervision announce deferral of Basel III implementation to increase operational capacity of banks and supervisors to respond to Covid-19", 27 March 2020

BCBS Press release "Basel Committee sets out additional measures to alleviate the impact of Covid-19", 3 April 2020

It works like a very fine "molecular knob" able to modulate the electrical activity of the neurons of our cerebral cortex, crucial to the functioning of our brain.

Baltimore Orioles outfielder and first baseman Trey Mancini revealed Tuesday he is in the midst of six months of chemotherapy as he fights Stage III colon cancer.

The Korean baseball league opens its delayed season Tuesday in empty stadiums across South Korea and Canada's Jamie Romak, along with the rest of the league's players, are getting used to a new baseball normal that includes rules against high fives and spitting, daily temperature checks and the absence of fans.

Visit our four downtown locations where you can taste euphoria in our creative and delicious all natural ice-cream, yogurts and sorbets!

Bike the Sites of the Nation's Capital! It's the fun and easy way to get to get up close and personal with Washington's landmarks

Double Decker Tours offers the best experience. Our Double Decker buses have the most visible sights like US Capitol, White House, Museums and Memorials

Discover the real George Washington through his restored home and gardens, over 1,000 artifacts, and an immersive Revolutionary War "snow" experience

Voted "Washington's Best Tour" by Washingtonian Magazine. See the best of Washington aboard on our 2 hour tour with live narration

Take a bus tour to the sites of movies and TV shows. Your guide will entertain you as you visit over 30 locations used in West Wing, The Exorcist and more

Get up close to the DC sites on a Segway! Segs in the City offers daily guided 1 hour and 2 hour Segway tours and rentals. Join the fun!

The Best Museum Experience of All Things Washington, D.C. Experience the stories behind the people and events of the Nation's Capital.

A dead body was found along the CP Rail line near Molson, Manioba, RCMP say.

The union that represents thousands of Manitoba workers is asking what safety protocols were in place when a construction worker was killed after a trench wall collapsed on him earlier this week.

They're not necessarily treating sick patients in hospitals, but a number of Manitoba-based scientists are working long hours and facing incredible pressure to battle the novel coronavirus from their labs and research facilities.

Legal experts and leading scientists have teamed up with Creative Commons to create the Open COVID Pledge to help speed up the battle against the coronavirus pandemic. The Pledge gives broad permission to anyone to use intellectual property not otherwise accessible to the public, and generally replaces the need for any other license or royalty agreement.

Contact tracing is a big job, like trying to drain an ocean with a teaspoon. It involves finding people who have been exposed to the coronavirus and testing them to determine if they are infected or are carriers. Public health officials then can take necessary steps to prevent the virus' spread. It's a perfect fit for CRM, and Salesforce's core technology is coming to the forefront.

Oracle and Zoom just entered a deal that for once is more about technological audacity than about dollars -- a partnership to host Zoom on Oracle Cloud Infrastructure. In just a few months -- basically since the beginning of the novel coronavirus pandemic -- Zoom has seen demand for its service grow from about 10 million daily meeting participants to more than 300 million.

Prices on P.E.I. for gas, heating oil and diesel all took a big jump upwards Friday.

With opposition parties continuing to call for the legislature to be convened, P.E.I. Premier Dennis King says that opportunity for them to examine the government’s spending is coming.

All of P.E.I.'s 27 confirmed cases of COVID-19 are now considered recovered, said Chief Public Health Officer Dr. Heather Morrison.

Sheldon Lavers didn't leave his window for hours Friday night as police negotiated with a man next door who they believed had a gun and was smashing items inside.

Former Michigan Gov. Rick Snyder is headed to Harvard University to teach, study and write on subjects related to state and local government.

Former Michigan Gov. Rick Snyder says he won’t continue with a fellowship at Harvard University following backlash over his role in Flint’s water crisis.

The Flint Registry is holding in-person enrollment across the city of Flint all week. Officials are trying to track the health of residents made ill by the Flint Water Crisis.

Planned Parenthood's president says many states used the pandemic to restrict abortion access; Director Eliza Hittman on her new film about abortion, "Never Rarely Sometimes Always"; Science journalist Tanya Lewis on drugs being tested to treat COVID-19.

In a Democracy Now! exclusive, singer-songwriter Fiona Apple discusses her acclaimed new album, Fetch the Bolt Cutters; and Native American activist Eryn Wise of Seeding Sovereignty talks about land acknowledgment and COVID-19 in Indigenous communities.

President Trump lashes out at the U.S. Postal Service. His attacks could threaten voting by mail; We look at the impact of the coronavirus on schools, universities, students, parents and teachers, and who is at the table to shape what happens next.

French economist Thomas Piketty on how the economic crisis triggered by the pandemic may be a catalyst to address global inequality; longtime WHO adviser Lawrence Gostin on Trump's order barring states from closing meatpacking plants with virus outbreaks.

Workers call for a general strike on May Day; Joe Biden denies Tara Reade's sexual assault allegations. We speak with her neighbor from the 1990s who says Reade told her the story decades ago; Protesters demand more COVID-19 relief & tests in Puerto Rico.

As meat plant workers get sick and die from COVID-19, workers protest conditions, and LULAC calls for Meatless May Mondays; McConnell seeks to protect corporations from liability; Dr. Richard Levitan says patients unknowingly suffer oxygen deprivation.

As the Navajo Nation suffers the third most COVID-19 cases, we talk to the partner of a 28-year-old victim and doctors treating patients; 80 percent of prisoners at Marion prison test positive; Prof. Ruth Wilson Gilmore on the case for prison abolition.

As President Trump starts to reopen the country, Pulitzer Prize-winning science writer Laurie Garrett predicts the pandemic will last at least 36 months; Did a former Green Beret mastermind a failed coup attempt in Venezuela? Two Americans were arrested.

We get an update from Moscow on the pandemic in Russia, which has the second-highest infection rate in the world; We look at who gets access to drugs like remdesivir being developed by pharmaceutical giant Gilead, which is poised to make massive profits.

Two white men are charged with the murder of Ahmaud Arbery, more than two months after his killing; The pandemic hits African Americans hardest. We speak with NYT Magazine's Linda Villarosa; Dr. Leana Wen on what the U.S. faces as states begin to reopen.

Remarks (delivered via videoconference) by Mr John C Williams, President and Chief Executive Officer of the Federal Reserve Bank of New York, at the Economic Club of New York, New York City, 16 April 2020.

Quebec Health Minister Danielle McCann says the government is evaluating whether to allow the world figure skating championships to go ahead next week in Montreal.