How do you heat shock bacteria?
How do you heat shock bacteria?
When time is up, heat shock the cell and plasmid mixture by placing it in a water bath at 42˚C for 30 seconds. Immediately after taking the tube out of the water bath put it on ice and add 450μL of media. Place it in a shaking incubator for 37°C for 1 hour at over 225 rpm so that the cells can recover.
What is the purpose of heating the tubes to 37 ̊C?
The use of the 37C incubation is to allow the cells to begin growth and ensure that the antibiotic resistance casette on the plasmid has some time to be expressed before putting them on a plate with antibiotics.
What is the basic principle of bacterial transformation?
Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. It was first reported in Streptococcus pneumoniae by Griffith in 1928. DNA as the transforming principle was demonstrated by Avery et al in 1944.
What happens if you heat shock too long?
Incubate for 10 minutes on ice. Heat shock cells for 45 seconds at 42C in a heat block. (Do not go over 45 seconds!) You can kill bacteria by keeping them at high temps for too long.
Why is it necessary to return the tubes to ice after heat shocking?
The heated mixture is then placed back on ice to retain the plasmids inside the bacteria. Many cells do not survive the rapid temperature change but enough maintain integrity to keep the plasmid and, when medium is added, recover and divide.
Why do you incubate at 37 degrees Celsius?
Mammalian cells operate optimally at 37 oC – molecular kinetics of enzymes and their substrate increase as the temperature increases, meaning a greater number of biochemical reactions can occur. Lower temperatures are less efficient.
Why is the incubator oven set for 37 degrees C?
The air in the incubator was kept at 37 degrees Celsius, the same temperature as the human body, and the incubator maintained the atmospheric carbon dioxide and nitrogen levels necessary to promote cell growth.
What is DH5?
DH5-Alpha Cells are E. coli cells engineered by American biologist Douglas Hanahan to maximize transformation efficiency. They are defined by three mutations: recA1, endA1 which help plasmid insertion and lacZΔM15 which enables blue white screening.
