Experiment 1, Day 3

Question 1

What is the formula for “Transformation Efficiency”?

Answer 1

of colonies/ # of cells started with

Question 2

Solution: Resuspension Solution

Why/What? Ingredients:

Answer 2

Glucose- Increase osmotic pressure outside cell.

Tris-Buffering agent, maintains pH; solubilizes DNA; protects DNA from degradation.

EDTA- Chelating agent—>protects DNA from degradation by binding divalent cations necessary for DNase enzyme activity.

Question 3

Solution: Lysis Solution

What/Why? Ingredients:

Answer 3

NaOH- Helps cell lysis; denatures DNA into single strands (increase pH)

SDS- Detergent; breaks apart and solubilizes lipid membrane and proteins

Question 4

Solution: Neutralization Solution

Why/What? Ingredients:

Answer 4

Potassium Acetate- Dissociates K+ and CH3COO- (acetic acid); acid neutralizes the pH, allowing DNA strands to renature; K+ neutralize negative charge on the PO3- groups on nucleic acids, making less hydrophilic and less soluble in H20.

Question 5

Solution: Wash Solution

Why/What? Ingredients:

Answer 5

100% Ethanol- Helps remove salts and SDS (but keeps DNA on column)

Question 6

Solution: Elution Buffer

Why/What? Ingredient:

Answer 6

Tris or H20- Releases DNA from column; buffer helps stabilize DNA

Question 7

Basic steps of Alkaline Mini-Prep kit

Answer 7

1. Take your tube of transformed cells, pellet them by centrifugation
2. Remove the supernatant and then resuspend them in a resuspension solution that contains RNase A.
3. Then you will add Lysis solution to break open the cells and release their contents.
4. Neutralization solution is added, contains acid that lowers the pH, precipitates proteins, and causes the DNA to renature.