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 9, April 2020. By allowing banks to run down some of their buffers, policymakers are sending a strong signal about their resolve to lessen the economic fallout from the pandemic. Such prudential measures complement the main policy levers: monetary and fiscal instruments. To avoid a reduction in credit to the real economy, authorities need to ensure that banks have the capacity and willingness to make use of the flexibility afforded by the buffer release. Payout restrictions on banks and risk-sharing between banks and the public sector will be key. For banks to continue playing a positive role in the supply of funding during the recovery, they should maintain usable buffers for a long period, as losses from a severe recession will take time to materialise.

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

The number of people evaluated for signs of stroke at U.S. hospitals has dropped by nearly 40% during the COVID-19 pandemic, according to a study led by researchers from Washington University School of Medicine in St. Louis who analyzed stroke evaluations at more than 800 hospitals across 49 states and the District of Columbia.

As Nova Scotians gazed up at the Canadian Forces Snowbirds soaring through the clouds last weekend, Matt MacKenzie was looking down and feeling the weight of successive tragedies that had enveloped his home province. 

Live professional baseball games could be televised in the United States as early next week, with South Korea's Yonhap News Agency reporting Monday that ESPN and the Korea Baseball Organization are nearing an agreement.

Baseball Hall of Famer Andre Dawson spoke to CBC's John Northcott about the realities of running a funeral home during the pandemic.

With Mother's Day only a few days away, businesses are coming up with creative ways to help your family celebrate. Everything from brunch in a box to colourful bouquets and home delivered chocolate.

In this uncertain and increasingly homebound era, customers are turning online for everything from groceries to cat food. It's more important than ever to make sure that websites are optimized for the highest-possible customer engagement and conversion. E-commerce optimization is one game that companies struggling for market share in the midst of a pandemic must play.

Women on P.E.I. are having a harder time holding onto their jobs than men in the COVID-19 pandemic, which runs contrary to the national trend.

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

Nearly 5 ½ years ago, the water source for the city of Flint was switched in an effort to save money. A disaster followed resulting in deaths, illness and mistrust of government. Tuesday at 10:00 p.m. on WKAR-TV, the documentary series Frontlin e examines the crisis by bringing to light conversations and documents never seen on television before. We spoke with Abby Ellis, the Michigan native who is the film’s director.

Robbyn Plumb tried to balance a career in the public service with looking after two kids, one with special needs. When the pandemic hit she finally hit a wall and decided to stop working. She writes about how hard it is for parents trying to do it all during COVID-19.

Music Division Archivist Morgen Stevens-Garmon reflects upon American playwright, screenwriter, and librettist Terrence McNally, who died of complications due to COVID-19 on March 24, 2020.

Canadian basketball players Dylan Ennis and Kevin Pangos, who both play in Liga ACB, opted to stay and weather the COVID-19 pandemic in Spain.

Hobbies during quarantine: How they can help us in a pandemic  Today.com

From stage to sofa: NAC brings local arts to homes in new livestream initiative  Connected to India

Young singers bring old Tamil gems to new gen  Times of India

"This video footage clearly shows why La Loche Saskatchewan won't be opening up so soon," says Rita Briggs.

The leader of a First Nation surrounded by oilsands development is frustrated by the Alberta Energy Regulator's decision to suspend a wide array of environmental reporting requirements for oilsands companies.

Manitoba health officials have an agreement with First Nations leaders to track and share COVID-19 data, but the public may never know specifics of what the unique agreement yields.

The federal government is looking to hire paramedics who can fly up to remote First Nations in case there’s a surge of COVID-19 cases, and officials say it’s evidence of a different approach to Indigenous health care than during the H1N1 outbreak.

The Canada-wide chain will introduce a mandatory mask policy on May 11, claiming customers and employees want a policy more in line with how Asian countries have handled the COVID-19 crisis.

As part of the province's approach to curbing the COVID-19 pandemic, there are a number of rules in place that people have to follow. Kitchener and Waterloo bylaw officials offer some insight on what's allowed and what's not.

The COVID-19 pandemic might be wreaking havoc with Brian Jones' off-season conditioning but it's certainly enhancing his musical skills.

As a farmer, spring means a fresh start, a chance for new life, hope and opportunities.

This collection of songs heard on Saskatoon Morning will be updated weekly.

Vancouver police are investigating an incident where a man attempted to lure a child to his car in the city’s West Point Grey neighbourhood on Wednesday.

In the middle of a pandemic, who wants to see politicians engage in traditional games of partisan finger pointing and over-the-top attacks?

An idea that began when a classicist went to a brewery to sip beers and ponder the history of hops has brought to life an ancient ale.

The media is allowing disinformation to appear as news.

The eerie inhumanity of Donald Trump.

Karla Bell was outside an Orangeville hospital waiting to start her nursing shift when she heard a cry for help from a car in the parking lot. She ran up to find a woman in labour in the passenger seat. Natalie Kalata tells us the incredible story of how this baby was born.

Approximately one out of every seven Ontarians who were working before the coronavirus pandemic hit the province have now lost their jobs, according to Statistics Canada's latest national labour survey.

Lindsay Forsyth Brochu thought by now she'd have the double-lung transplant she's been waiting for. But she had the misfortune being put on the waitlist the day after most surgeries were suspended in Ontario due to COVID-19.

Paramedics who come back from COVID-19 related calls have to remove and either discard or wash their PPE. They are getting help from students called doffing assistants.

With proms cancelled across Canada, an online resource for kids called the Student Life Network has organized a national virtual prom.

Making a film when you can't leave home means being creative — with everything from the scenery to your cast. C.J. Bernauer talked to Windsor Morning host Tony Doucette about his new short horror film, called 'You're It.'

As truckers move vital supplies such as food, hand sanitizer and ventilators during the COVID-19 pandemic, they're pleading with governments to allow more truck stops to reopen so they can get meals, showers and rest. Social media groups and individual businesses are doing their part to thank truckers.

Justin Young and Justine Manuel in Kamloops, B.C., shaved off their eyebrows as extra incentive to stay home and avoid socializing.

A 23-year-old man has been granted bail, after being charged in the death of a Saskatchewan farmer with a wife and two kids whose body was found dumped north of Calgary.

A Toronto law firm has filed to launch a class action lawsuit against a Niagara Falls long-term care home where 18 residents have died from COVID-19, saying the home had a pub night in the midst of the pandemic.

Quantifying RNA is an important and necessary step before most RNA analysis techniques. Methods for quantifying RNA can be classified into two categories: ultraviolet (UV) spectrophotometric methods, which are based on the absorption spectra of the purine and pyrimidine bases; and fluorescent dye-based methods, which measure the fluorescence intensity of dyes that selectively fluoresce when bound to nucleic acids. If the RNA sample is pure (i.e., without significant amounts of contaminants such as proteins, phenol, agarose, or other nucleic acids), UV spectrophotometric measurement of the amount of UV irradiation absorbed by the bases is simple and accurate. However, if the sample contains significant quantities of impurities or if the concentration of RNA is very low, it is better to use fluorescent dye-based methods. An overview of spectrophotometric and fluorescent dye-based RNA quantification methods is given here, as are several options for storing purified RNA preparations. Proper storage of RNA samples is important; it can help minimize RNase contamination and consequent sample degradation.