first, include an abstract on report. I will include all the information and you can put that together in your own
words.
Objective: to understand the structural characteristics of DNA that are responsible for its viscosity, perform
viscosity tests, to illustrate that viscosity of DNA can be reduced by shearing or denaturation; to understand
why DNA is viscous in solution but not in intact cells
DNA is Polymer consisting of repeating nucleotide units
Each nucleotide =nitrogenous base, deoxyribose sugar, and inorganic phosphate
DNA is double helix and rigid
Four bases = A, C, T, G Strands are held together by hydrogen bonds (noncovalent)
Adenine pairs with Thymine
Guanine pairs with Cytosine
A and G-purines
T and C-pyrimidines
Experiment 1: Visocity Test
Viscosity-resistance of a substance to flow. DNA is viscous because of its length and its double helix.
We will perform the viscosity test of the suspension (Unlysed cells) and cell lysate using Pasteur pipet and
the timer.
Use the same pipet for each measurement, no need to clean it!
Wear protective eye wear!!
The tip of the pipet is very sharp!
We will use 10% sodium dodecyl sulfate (SDS) and 10N sodium hydroxide (NaOH) to break open the cells
(will be added by the instructor).
How SDS works?SDS +NaOH=lysis buffer
SDS solubilizes cell membrane, binds to both hydrophobic (membrane) and hydrophilic (non-membrane)
proteins.
NaOH helps break down the cell wall
SDS also denatures most of the proteins in the cell.
DNA that has been sheared (cut or broken up) is less viscous than intact DNA.
Shearing of DNA reduces its length.
Experiment 4-2-Mechanical shearing of DNA
We will be using same 4 ml of the cell
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lysate for all 5 shearing passes.
Heating DNA denatures it (breaks apart the strands of the double helix). What bonds are being broken?
Single-stranded DNA is less viscous than double-stranded
Experiment 4-3
Don’t forget the put boiled DNA on ice!!!
Use 2 ml of the cell lysate
DNA can be precipitated by dehydration in alcohol solutions
Precipitated DNA can then be spooled using a glass rod
Experiment 4-4
4ml of lysate+ 8ml ethanol
Will observe white threads of DNA
Sample Solution
