Fundamental Cell and Molecular Bio Lab Practicum 1

Total Magnification

Objective Lens + ocular lens= total magnification

What does a Kim wipe clean?

ocular and objective lens

How to carry a microscope

One hand on base, other on arm (two hands at all times)

What lenses to use?

Coarse focus only at 40x, and fine focus knob on all of them

Micropipettes vs. Serological Pipettes

Micropipettes measure 1-1,000uL (Gilson and Fisherbrand pipetteman)

Serological Pipettes 1,-50mL

Size range of a microscope at scanning objective

At 40x, about 5mm

Average size of a yeast cell

3-5 micrometers

Highest power objective?

The oil immersion is the highest power objective (1000x) and oil is needed to help zoom in because it is so close

Successful serial dilutions

Each successive dilution is 1/2 the value of the previous well

What role does Sodium dodecyl sulfate (SDS) play in DNA isolation?

SDS disrupts the lipid membrane and denature the proteins because of its polar and nonpolar ends, it lyses the cell

Role of phenol and chloroform in DNA isolation

they are protein solvents and will denature the proteins, phenol is only partially soluble so it forms a layer on the bottom with the proteins, lipids, cell lysate (While nucleic acids will be on the top layer in the aq phase)

Role of ethanol/salt in DNA isolation

Ethanol will concentrate the nucleic acid, and precipitate it turning it into a solid again

How is the RNA separated from the DNA?

The RNAse digests the RNA concentrating the DNA

How UV absorbance at 260nm and 280nm is used to determine DNA purity and the significance of the 260/280 ratio.

absorbance of DNA is at 260nm, and OD260/OD280 should equal 1.8, 260 is the peak

Purpose and role of Centrifuge

Centrifuge uses density and gravity to separate cellular components, it spins super fast causing the heavier and more dense materials to go to the bottom and creates layers

Purpose of a fume hood

Fume hoods prevent hazardous chemicals from mixing with the general room air.

Understanding how colchicine disrupts cell division

Colchicine inhibits the polymerization of microtubules which stops the spindle formation which draws the chromosomes in cell division

The effects of trypsin on breaking down cell structures in chromosome analysis.

Trypsin digests the proteins and they are washed away by the chilled PBS

The importance of using testicular tissue in karyotyping

Due to it's high rate of cellular division, when cells are dividing the chromosomes are most visible, additionally meiosis is happening at a high rate in the formation of gametes which are necessary for zygote formation

Techniques for preparing chromosome spreads

Thermal lysing: warming the slides, and mechanical lysing is dropping the solution from high above to break open the cellular components

How are chromosomes identified

length and banding patterns

Conversion between units

micro (10^-6) in comparison to L, milliliters (10^-3) in comparison to L

Volume of a cylinder

V=πr²h

Properties of phospholipid bilayer and what can pass through it

polar heads (like water, usually charged), nonpolar tails, double membrane, only small nonpolar molecules can pass through easily (and through simple diffusion) (C, O), H2O through concentration gradient, through facilitated diffusion larger molecules like glucose, charged ions, and large polar molecules can pass through

Osmotic stages

Isotonic (equal levels of solute and water on each side), Hypotonic (low amounts of solute outside membrane so water comes into the cell making it burst), hypertonic (higher solute outside, water leaves making it shrivel )

Independent vs Dependent Variable

An independent variable is the variable that is changed or controlled in a scientific experiment, the dependent variable is the one effected and observed (dependent on x, independent on y), x is usually time

Diameter of scanning objective

40x has about a 5mm field of view

Average size of a yeast cell

3-5micrometers

Use of oil immersion objective

Oil immersion is the highest power objective at 1000x, oil helps to view things clearly and zoom in

How are we able to predict the absorbance values based in dilution factors?

The more dye, the higher the absorbance value, the graph should be a straight line because the serial dilutions should be very exact

How to make sense of the DNA 260/280 ratio

DNA's absorbance is 260nm while proteins is at 280, basically DNA is measured at 260 so a high amount should be at the 260 value, and we don't want a high amount at 280 because then that would indicate that there is a significant amount of proteins being measured (there still will be some because they can't fully be separated, but we test the DNA purity by dividing OD260/OD280 to get 1.8)

What molecule is amphipathic when extracting the DNA and what is its purpose?

The SDS is amphipathic so it has both a polar and non polar side basically taking apart the phospholipid bilayer of the cell (like dissolves like)

Read this caliper measurement

1.21

The principles of Benedict's test for detecting glucose and interpreting results

Blue (no glucose) red (high glucose)

The use of iodine for detecting starch and how to enhance faint results

Dark blue is positive for starch and yellow is negative