Cell Bio Lab Exam II

Electrophoresis

Is a method whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to an electric field

1- Strength of the field

2- Net charge, and size of the molecules

3- Shape of the molecules, and ionic strength

4- Viscosity, and temperature of the medium in which the molecules are moving

What does the electrophoresis migration rate through the electrical field depend on?

HINT: there are four things

In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

What is electrophoresis?

What are the types of electrophoresis?

Paper – small molecules and amino acids

Polyacrylamide gel – proteins and nucleic acids

Agarose gel – very large proteins and nucleic acids • Starch gel – proteins and nucleic acids

Gel electrophoresis can be divided into two categories:

HINT: there are two dimensions; one contains three and the other contains one

One dimension

• SDS-PAGE

• Native-PAGE

• Isoelectric Focusing (IEF)

2. Two dimensions

• 2D-PAGE

Native gel electrophoresis

separation is based on size, charge, and shape

Isoelectric focusing electrophoresis

separation is based on isoelectric point

SDS-PAGE electrophoresis

separation is based on size

2 dimensional gel

based on two separations

first separation is based on isoelectric point and second separation is based on size through SDS page

What is the gel matrix composed of?

agarose or polyacrylamide

Is a cross-linked polymer of acrylamide

polyacrylamide

Acrylamide

is a potent neurotoxin and should be handled with care!

Lab procedure for lab 5

1. Set the precast gel based on the video. https://www.youtube.com/watch?v=XnEdmk1Sqvg

2. Mix 5uL protein sample with 5uL loading buffer.

3. Heat the sample at 95oC-100oC for 5min, then load into the well.

4. Run the gel at 150V until the front dye reach the bottom.

5. Stain the gel with 100mL Coomassie stain solution.

6. Destain the gel with 100mL destain solution.

Electrophoresis

A separation technique that exploits the differential migration of molecules in an electrical field depending upon their size and charge

Variables that are exploited in the electrophoresis are:

polarity

voltage

length of application

current

What is the cross-linking agent we used in lab 5 ?

N,N methylene bis acrylamide

The pore size determines what in lab 5?

rate of migration

The pore size decreases with increased what ?

Increased T

The gelling process requires what two things ?

what two things do we use for lab 5?

initiator and catalyst

initiator is ammonium persulfate

catalyst is TEMED

The top of the electrode is positive or negative ?

Negative cathode

The bottom of the electrode is positive or negative?

Positive cathode (anode)

What stays constant in lab 5?

what is our main concerns when running gels?

current

voltage

power constant

heat build-up and its effects on the proteins

Polyacrylamide gels

Have smaller pores than agarose, therefore high degree of resolving power.

Can separate DNA fragments which range in size from 10- 500 bp.

DNA fragments that differ in size by one nucleotide can be separated from each other.

Polyacrylamide gel electrophoresis is also used to separate protein molecules.

Protein electrophoresis

Separate proteins based on

Size (Molecular Weight - MW)

Allows us to • characterize • quantify • determine the purity of the sample • compare proteins from different sources •And it is a step in Western blot

SDS page

The purpose of this method is to separate proteins according to their size, and no other physical feature

The end result of an SDS has two important features: what are they?

1. All proteins contain only primary structure

2. All proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field

______________ is the most frequently used cross-linking agent for polyacrylamide gels

Bisacrylamide

Smaller proteins will move through the gel _________ while larger proteins move at a ____________ pace

Faster; Slower

________ are ionic substances that become partially _________ in water dissociated

Buffers; dissociated

What pH level are the following tanks and buffers?

tank buffer

stacking gel

separation gel

8.30

6.90

8.48

SDS PAGE: separates proteins primarily based on their molecular weight

In lab 5 which protein electrophoresis did we complete ?

The further down a gel protein has gone the smaller the __________ __________ is

molecular weight

How to calculate %T

%T = total percentage of acrylamide (monomer) and bisacrylamide (cross-linker) per 100 ml of solution.

Found at the top of the gel and contains the wells. - Has lower %T (larger pore size), lower pH. - All of the protein samples line up and enter the separating gel at exactly the same time.

stacking gel

Separating gel

Has higher %T (smaller pore size), higher pH. - Proteins will separate based on size.

loading buffers

Tris-HCl,

SDS,

glycerol,

beta-mercaptoethanol,

bromophenol blue.

running buffers

Tris

glycine

SDS pH 8.3

DNA extraction id divided into six steps:

tissue homogenization

plant cell lysis in DNA extraction buffer

separation of DNA from other cellular components

DNA precipitation

DNA washing

DNA resuspension of downstream

Plant cell lysis includes what four things

Tris

EDTA

NaCl

SDS

B mercaptoethanol

what is the neutralization buffer in lab 6?

Bio 101 solution III

Nucleic acids and proteins have an absorbance maxima at ____nm and ____nm, respectively.

Pure DNA will have an A260/A280 ratio of ________-__________.

260-280

1.8-2.0

Lower 260/280 ratios usually indicate that a sample is contaminated by residual phenol, sugar, or the reagents used in the extraction protocol.

True or false

TRUE

Tris contains a mixture of ______ acid and its _______ ________ acid, acts as a buffer and increases permeability of cell wall

weak

conjugate weak acid

EDTA cations such as Mg 2+ and Ca2+ which reduces the _________ ___________- of DNase

enzyme activity

NaCl helps to remove proteins that are bound to the _______

DNA

SDS ___________ proteins which helps ______ ________ ________

linearizes

plant cell lysis

To get quality DNA what should be removed?

phenolics compounds

B mercaptoethanol is a strong _____ agent

reducing

Sodium acetate with isopropanol are used for _______ _________________

DNA precipitation

introns

non coding sequences and od not code for proteins

removed by RNA splicing as mRNA matures

Exons

are coding sequences and code for proteins

Principle of PCR is what three steps?

denaturation

annealing

extension

Agarose gel electrophoresis

is a method used to separate DNA fragments by size in a solid agarose gel which is a common way to check the PCR products

The rate of migration in lab 7 and 8 is proportional to what ?

size

DNA loading dye serves a few purposes:

coloring and to easily track your sample

loading dye is dense and keeps the DNA in your sample from diffusing away in the buffer

After gel electrophoresis, the DNA can be visualized under ______ due to the intercalating dye ethidium bromide or SYBR gold

UV

The PCR nucleotide mix in lab 7 and 8 is what?

dNTPs

The agarose gel electrophoresis has _________ resolving power than SDS page

Lower

The higher the concentration of agarose, the __________ the pore size

Smaller

Factors that affect DNA migration in lab 8 is what ?

agarose concentration

size of DNA molecule

DNA conformation

Applied voltage

In lab 8 what loading dye will we use?

bromophenol blue and sucrose

cDNA no introns is what bp

gDNA with introns is what bp

694

805

RT PCR is _______

PCR product is _________

cDNA

gDNA

Why is the size of the RT-PCR product different from that of the genomic PCR product?

intron exon structure

primer design

polyadenylation

PCR conditions

In PCR, what does annealing temperature depend on? what determines the extension time that should be used during PCR

Primer length and composition

Target DNA

polymerase being use in reaction