Lab 9 Dna Gel Electrophoresis

Front 1

Gel Electrophoresis

Back 1

Is a procedure to separate biomolecules such as DNA, RNA or proteins using a gel matrix made of agarose or polyacrylamide.

* Separation depends on the properties of the gel as well as the molecules to be separated.

Front 2

Gel Matrix

Back 2

Contains pores of a uniform size through which molecules can pass through.

Front 3

What does the size of the pores in gel matrix depend on?

Back 3

Gel material

Front 4

Which makes larges pores compared to polyacrylamide?

Back 4

Agarose

Front 5

Gel Concentration

Back 5

Determines the size of the pores

Front 6

T/F: The higher the concentration of the gel the smaller the pores will be.

Back 6

True

Front 7

T/F: Large molecules don't pass easily through the small pores.

Back 7

False: Large molecules pass easily through the small pores.

Front 8

What separates large molecule of nucleic acids DNA and RNA?

Back 8

Agarose gel

Front 9

What is polyacrylamide used to separate?

Back 9

Used to separate proteins, which are MUCH SMALLER than DNA and RNA

Front 10

Size of molecules

Back 10

Is the most important factor for migration through the gel matrix

Front 11

T/F: Larger molecules migrate more slowly as compared to smaller molecules.

Back 11

True

Front 12

Electric Current (3 points)

Back 12

Molecules require electrical current to move through the gel pores.

The higher the voltage, the faster the DNA moves

**High voltage also heats the gel and ultimately causes the gel to melt, denature the DNA, and decrease the resolution ( separation) of the DNA fragments.

Front 13

What are the three negative effects of Electric Current?

Back 13

**High voltage also heats the gel and ultimately causes the gel to melt, denature the DNA, and decrease the resolution ( separation) of the DNA fragments.

Front 14

Charge

Back 14

Affects the migration of molecules through an electrical field.

When current is applied, positive molecules (cations) will move towards the cathode (negative electrode) while negative molecules (anions) will move toward the positive electrode (anode).

Front 15

T/F: Molecules with the same size but different charge will DIFFER in migration

Back 15

True

Front 16

T/F: To separate molecules by size only the charges do not have to be the same.

Back 16

False: To separe molecules by size only, all the molecules must have similar charge.

Front 17

Why is water not used for preparing gels or used as running solution?

Back 17

Because it is not a good conductor of electricity.

Front 18

Running Buffer/ TAE buffer pH 8

Back 18

Contains electrolytes is used to endure the conductance of electricity through the gel

Front 19

What is the charge of ionized DNA and RNA molecules?

Back 19

Contain ionizable phosphate groups that are NEGATIVELY CHARGED at pH 8

Front 20

Do all DNA fragments have same charge?

Back 20

Not sure most likely yes

Front 21

T/F: DNA will move towards the cathode

Back 21

False it will move towards the anode

Front 22

Shape of molecule

Back 22

1. Can interfere with migration through the gel

2. Molecules of same size and charge differ in their migration when they vary in shape

Front 23

When is isolated DNA supercoiled?

Back 23

Uncut circular plasmid (undigested) is generally supercoiled and runs FASTER than linear DNA of the same size.

Front 24

When is nick and linear DNA observed?

Back 24

In a poor quality DNA uncut prep.

Front 25

What does the DNA loading concentrate contain?

Back 25

1. A blue tracking dye

2. Glycerol

Front 26

What does the colored blue tracking dye do?

Back 26

It has a very low molecular dye and runs ahead of the DNA fragments and tracks the progress of the colorless DNA through the gel.

Front 27

What does the glycerol do in the DNA loading buffer?

Back 27

Glycerol increases the density of the sample and facilitates it to fall easily into the wells and prevents it form floating away.

Front 28

DNA marker

Back 28

Contains a mixture of DNA fragments of known sizes is used to determine the size of the DNA in the samples.

Front 29

What is used to compare and analyze the gel observations?

Back 29

Digested (cut) and non-digested (uncut) DNA.

Front 30

T/F: Loading DNA in the smallest volume possible will result in sharper bands.

Back 30

True

Front 31

TAE (Tris Acetate EDTA)

Back 31

1. Provides better resolution of fragments >4 kb

2. Gel is covered with 2-3mm buffer

3. TOO much buffer decrease migration of DNA and distorts the DNA bands.

Front 32

What is the common technique to visualize the colorless nucleic acids?

Back 32

Use of Dyes

Front 33

What highly sensitive stain is used for visualization of nucleotide strands in agars or acrylamide gels?

Back 33

SYBR Safe DNA Gel Stain

Front 34

SYBR SAFE functions

Back 34

1. Binds with double stranded DNA to form a complex that fluoresces under UV light.

2. It can also bind to single stranded nucleotides strands (RNA)

Front 35

What is used to determine the size of the DNA fragments in the sample ?

Back 35

Estimated by comparing their sizes with the DNA marker.

Lambda DNA digested with Hind III enzyme is often used as a DNA marker for sizing and to approximate quantification of DNA.

Front 36

T/F: the smaller bands are visible when the concentration of the DNA is low.

Back 36

False: The smaller bands are invisible when the concentration of the DNA is low.

Front 37

Properties of normal DNA

Back 37

Normal DNA is super coiled and this tight shape helps the DNA to move very fast compared to linear DNA formed after digestion with restriction enzymes.

Front 38

What does over exposure to alkaline conditions during DNA isolation do?

Back 38

Can hydrolyze only one of the DNA strand to form nicked DNA.

Front 39

What does a nick in DNA?

Back 39

Act like hooks and slows the migration of the DNA through the gel

Front 40

T/F: Under optimal conditions, restriction enzyme cut DNA at specific restriction sites.

Back 40

True

Front 41

When does EcoRi acquire non-specificity and cut at other sites?

Back 41

1. If the ionic conditions are too low (below 50 mM)

2. pH is not optimal

3. If the concentration of glycerol is too high

4. If too many units of enzyme are added

Front 42

Gene Expression

Back 42

Refers to the entire process from transcription through protein synthesis.

While genes requires for survival are generally expressed continuously (constitutive), the expression of most genes is regulated or controlled by other molecules.

Front 43

Gene Regulation

Back 43

Is a process that allows mRNA and protein synthesis only when and where the encoded protein is required.

One method of regulation gene expression is to control the synthesis of mRNA (transcription step)

Front 44

Examples of Gene regulation

Back 44

1. Myosin in muscles for contracting

2. Amylase in salivary glands fo carbohydrate digestion.

Front 45

Promoter

Back 45

1.Is a DNA sequence upstream to the coding region of a gene to which the RNA polymerase binds to initiate transcription.

2. Each gene or a set of genes requires its own promoter.

3. When a gene is regulated, the RNA polymerase cannot initiate transcription unless the regulatory molecules are present or when the inhibitor of the promoter region is released.

Front 46

Regulation

Back 46

Is responsible for differentiating stem cells to function specific cells.

To turn on and off genes is commonly used in biotechnology

Front 47

Example of regulatory molecules

Back 47

In a stem cell, actin (protein) is not present. As a result, the stem cells do not have muscle phenotype (do not have appearance & function of muscle cells)

To become muscle cells, the stem cells must express actin.

Front 48

In pGLO transformed bacteria, L(+) arabinose & Ara C protein are required to…..

Back 48

Initiate transcription of the GFP gene

Front 49

Where are the several different cellar proteins required for function enclosed in?

Back 49

Inside the cell membrane

Front 50

What are the ways to open up (disintegrate the cell membrane? Protein extraction

Back 50

1. Enzymes

2. Chemicals

3. Stress (freeze thaw, homogenization

Front 51

T/F: The GFP protein is soluble and can be easily collected in the supernatant along with other soluble cellular proteins.

Back 51

True