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.

Our ongoing celebration of the Library of Congress National Book Festival continues with presidential scholar Annette Gordon-Reed discussing "The Hemingses of Monticello: An American Family" on the Special Programs stage at the 2015 Festival.

Our ongoing celebration of the Library of Congress National Book Festival continues with former Secretary of State Condoleezza Rice discussing "Democracy: Stories from the Long Road to Freedom" on the Main Stage at the 2017 Festival.

Even celebrity chef Julia Child said that the sleek appliance made mixing 'marvelous'

A new book explores the evolution of cartography throughout more than a century of commercial air travel

From gas stations to public libraries, these celebrity architect-designed buildings are worth a road trip

Press release about the Basel Committee publishing consultation paper on revisions to the credit valuation adjustment risk framework, 28 November 2019.

Press release: Basel Committee invites comments on the design of a prudential treatment for crypto-assets, 12 December 2019.

Markets Committee calls for wider adoption of global code of conduct for foreign exchange markets (Press release, 30 January 2020)

Basel Committee Press release "Basel Committee meets to review vulnerabilities and emerging risks, advance supervisory initiatives and promote Basel III implementationl", 27 February 2020.

Press release about Basel Committee assesses members' implementation of the Net Stable Funding Ratio and large exposures framework, 19 March 2020

Press release "Basel Committee coordinates policy and supervisory response to Covid-19", 20 March 2020

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

BCBS Press release "Basel Committee and IOSCO announce deferral of final implementation phases of the margin requirements for non-centrally cleared derivatives", 3 April 2020

BCBS Press release "Basel III monitoring results based on end-June 2019 data published by the Basel Committee", 8 April 2020

BCBS Press release "Basel Committee issues progress report on banks' implementation of the 'Principles for effective risk data aggregation and reporting'", 29 April 2020

Tissue engineering has a unique set of tools and technologies for developing preventive strategies, diagnostics, and treatments that can play an important role during the ongoing COVID-19 pandemic.

Classified released a music video for his song, Good News, this week featuring a pack of kids hanging out with seniors at a continuing care home in Enfield, N.S. It was filmed before the COVID-19 outbreak in the province.

Graduation is on hold for Dalhousie University medical student Manveer Bal, so he's decided to spend his newfound free time volunteering at the epicentre of the COVID-19 outbreak in Nova Scotia.

People in Truro, N.S., are reeling after the disappearance of three-year-old Dylan Ehler as the community tries to come to grips with another potential tragedy in the area.

Carmen Figueroa Sotelo has been interpreting all the news conferences for English audiences since the beginning of the pandemic in Quebec

Asylum seekers make up a large portion of the "guardian angels" Quebec Premier François Legault has praised in his daily COVID-19 briefings: the orderlies who have no guarantee they'll be allowed to stay in Canada.

The provincially appointed trustee of the English Montreal School Board says the Quebec government made the “right decision” when it postponed the reopening of Montreal-area elementary schools to May 25.

As the Montreal area continues to be the Canadian epicentre of the COVID-19 outbreak, anyone showing symptoms of the virus is being asked to get tested.

While the STM is strongly urging passengers to wear a face mask or face covering at all times, it won't be mandatory, said chair Philippe Schnobb.

Grim projections, travel rules and feeling safe while shopping at your local market — here's what's going on today.

Major League Baseball owners will discuss a length for the amateur draft next week and are likely to start the selections on the original date of June 10, a person familiar with the deliberations told The Associated Press.

Tampa Bay Rays' Blake Snell claimed champion status after defeating Lucas Giolito of the Chicago White Sox in the inaugural MLB The Show player league on Sunday.

A former groundskeeper for the Expos recently told the Montreal Gazette that Pete Rose, who played less than a full season with the team, routinely had an Olympic Stadium staffer cork his bats in 1984.

A Winnipeg School Division trustee who was suspended from the school board earlier this week does not plan on fighting the decision, she told CBC News.

"Recovery is not for the faint-hearted," says recovering addict Jeremy Raven. And sometimes, something as simple as a kind word may be the hand up that someone who is struggling needs, he says.

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.

With something like 380 websites being created every minute, building an e-commerce shop can be a daunting task. It's easy to look at all of the success stories out there and think, "How could I possibly be that lucky?" However, the opportunity for growth in e-commerce is not slowing down. E-commerce sales are projected to grow 85 percent from their 2019 totals by 2022.

Merchants soon will be able to sell products on Google Shopping at no charge. Previously, they had to pay per click, but the cost was not fixed. There was no minimum, but they had to set a maximum for ad spend and Google would stop displaying their ads once the maximum was reached. Starting next week, search results on the Google Shopping tab will consist primarily of free product listings.

Google is integrating its Google Meet videoconferencing application with Gmail, and it already appears as an option in some users' accounts. It is making the service available to everyone for free in the coming weeks, on the Web and through mobile apps for iOS and Android. Users will be able to start or join Meet videoconferences from within Google Calendar as well.

Sign makers in P.E.I. have been busy since the province announced its plans to ease back COVID-19 restrictions, as businesses are ordering signs and decals ahead of reopening.

The craft brewing industry in P.E.I. is suffering, with sales down from about 40 to 80 per cent.

Artists from Michigan and around the world are painting 50 murals in Flint to refocus the city's image on art rather than the lead-tainted water crisis.

Voters have elected a new mayor in Flint, Michigan, where recovery from a lead-contaminated water crisis remains a major issue.

OK, you’re gonna hear a lot about Joementum.

Almost every movie reminds Trump of … something it’s not.

The formative moment for a new generation.

Statement by Mr Ignazio Visco, Governor of the Bank of Italy and Governor of the Constituency of Albania, Greece, Italy, Malta, Portugal, San Marino and Timor-Leste, at the 101st Meeting (virtual) of the Development Committee (Joint Ministerial Committee of the Boards of Governors of the Bank and the Fund on the Transfer of Real Resources to Developing Countries), Washington DC, 17 April 2020.

Contribution by Mr Fabio Panetta, Member of the Executive Board of the European Central Bank, published by Politico on 21 April 2020.

Ottawa police have arrested a 15-year-old boy in connection with January's shooting. He's charged with first-degree murder and attempted murder.

The soil is ready to be tilled and seeds wait to be planted at community gardens across Ottawa after the province reversed a decision declaring them off limits during the COVID-19 pandemic.