1. This particular strain of E. Coli has been made \"competent\" by Calcium Chlo
ID: 202631 • Letter: 1
Question
1. This particular strain of E. Coli has been made "competent" by Calcium Chloride Treatment. Describe what it means to be "competent." (1 pt 2. In step 5, why should you not vortex the cells? (1 pt) 3. What is the reasoning behind the steps performed in steps 6 and 7 in the protocol? (1 pt) . Calculate the ug of DNA that will be transformed, when 50 ul of E. Coli Cells/ media is plated (this is referring to step 11 in the protocol. Show calculation (1 pt) 5. Predict whether or not each transformation plate will give rise to colonies (Fill in yes or no in the table DNA No DNA control below) (3 pts) LB-Agar Ampicillin/Chlorampenicol LB-Agar LB-Agar Ampicillin Plasmid DNAExplanation / Answer
1. Competent: Competence is the property of a cell to take up naked DNA. Some cells are naturally competent or can be made competent by various treatments for example Calcium chloride treatment.
2. Overtaxing can damage the Plasmid DNA and cells.
3. Heat shock to bacterial cell: When we keep it on ice, Calcium cations shows interaction with negatively charged DNA (neutralises the negative charge on DNA) and bacterial membrane is also stabilised. Calcium ions also coated on bacterial cell surface so DNA can easily attach on bacterial cell. After this bacteria cells are transferred to 42ºC in water bath, which gives heat shock for few seconds. This heat shock changes membrane fluidity and allows DNA molecule to enter the cell. Again cells are transferred to ice immediately so that membrane pores will be closed to allow DNA to remain inside the cell.
4. Amount DNA (µg) = Concentration of DNA (µg/µL) x Volume of DNA (µL)
Amount DNA (µg) = 10-3 µg (1 ng) x 1µL
Amount DNA (µg) = 10-3 µg
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