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Recombinant protein expression was maintained by addition of methanol to a final concentration of 0.5% v/v every 24 h. 3 h after the last methanol addition, cells were harvested by centrifugation and resuspended in 250 mL cold breaking buffer.
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"Cold breaks down the germination inhibitors in the seed.
There was a time a few years back where I saw a homeless man in a T-shirt and jeans on a terribly cold day in winter and I was horrified by how cold he was, and the fact that he had no jacket to shield him from the cold, broke my heart.
In fact, responding to a call for such a "cold" break-in can be costly for the Oakland Police Department.
When the cold breaks, about all anyone wants to do is strip off as many layers of clothing as decency allows and bask in the warmth for a few precious weeks.
In a moving scene late in the book, Lia's mother, a cardiac surgeon, portrayed as compulsive and cold, breaks down in tears, and we finally see her pain and love and confusion.
The cell pellets from 1 ml culture were resuspended in 500 μl of ice-cold breaking buffer (50 mM sodium phosphate, 1.0 mM PMSF, 1.0 mM EDTA, 5% glycerol, pH 7.4).
The immunization community also must move beyond the never-ending cycle of gap/barrier/bottleneck analyses that launch every initiative, identifying long lists of problems (for example, "cold chain broken," "supervision weak") and making unhelpful, anodyne recommendations ("fix the cold chain," "strengthen supervision").
"I always thought I wouldn't get a generator, but after four nights of trying to sleep in the freezing cold I broke down," he said.
She told me to scatter them over bare ground in February -- better yet if it had snowed -- because the seeds need cold to break their dormancy.
So during the association's annual conference in Toronto, which ended Wednesday, a little cultural cold war broke out among members over what are known as independent libraries in Cuba.
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