Mol Bio - Pio

Normal range for A260/A230 ratio

2.0-2.2

Decreased A260/A280 ratio interpretation

Increased protein

Increased ratio of A260/A280 interpretation

Residual phenol or Increased RNA contamination in DNA samples

Detection, comparison, or identification of nucleic acid strands based on size/length, secondary, and tertiary structures as accomplished through electrophoresis.

Nucleic acid resolution

In electrophoresis, DNA fragments migrate towards _

Anode (positive electrode)

Relationship of Fluorescence detected with the concentration of target DNA/RNA

Direct

Three formats of electrophoresis formats

1. Tube gels

2. Slab gels (horizontal or vertical)

3. Capillaries

Most frequent electrophoresis format used

Agarose (horizontal)

In agarose gel electrophoresis, if the pore size/space needs to be bigger, do we need increased or decreased concentration (of the gel)?

Decreased concentration

Relationship of pore size/size of the fragment and gel concentration (Agarose gel electrphoresis)

Indirect

Concentration range needed for agarose gel electrophoresis

0.5-5%

Identify the electrophoresis format:

• best used when resolving ssDNA and very small DNA fragments

PAGE

Identify the electrophoresis format:

• vertical oritentation

PAGE

Identify the electrophoresis format:

• pulses of current are applied in alternating dimensions

• pulses can move in different directions

PFGE

Identify the electrophoresis format:

• used when resolving very large pieces (50-250+ kbp)

PFGE

PFGE stands for _

Pulse Field Gel Electrophoresis

PAGE stands for _

Polyacrylamide Gel Electrophoresis

Identify the electrophoresis format:

• fused silica

Capillary electrophoresis

Identify the electrophoresis format:

• automated detection

• faster run time

Capillary electrophoresis

These components control the pH and carry the electrical charge in gel electrophoresis

Buffers

Relationship of buffer concentration and buffering capacity (increased electrical conductivity)

Direct

Least efficient isolation method of DNA

Centrifugation

These type of buffers are IDEAL for DNA electrophoresis

Tris buffers

Identify the Tris buffer:

• can sequester Mg

Tris borate EDTA

Identify the Tris buffer:

• inhibits enzyme based reactions

Tris borate EDTA

In Density gradient, the sample is found at the top or bottom of the well?

Bottom of the well

Relationship of voltage and resolution if gel electrophoresis

Indirect; if TOO LOW, band may become hazy

How many volts per cm length may used as a starting point when determining the optimal voltage in gel electrophoresis

5-10 volts per cm

Curved bands in gel electrophoresis are caused by increased or decreased voltage

Increased voltage

If gel electrophoresis takes longer, the resolution increases or decreases

Increases resolution

Ethidium Bromide color

Orange

Oldest fluorescent dye

Ethidium bromide

Identify the detection stain:

• only UV light can visualize this

Ethidium bromide

What stain is more sensitive, Ethidium bromide or SYBR stains?

SYBR stains

Which is more sensitive, SYBR stains or GelGreen and GelRed

GelGreen and GelRed

Most sensitive of the detection stains

Silver stains

TRUE OR FALSE: Silver stains cannot be incorporated into the gel or loading buffer

TRUE

Restriction sites are how many base pairs long?

4-8 bp

Restriction sites were observed in what organisms?

Bacteria

Type of cut that has NO OVERHANGS

Blunt end restriction endonuclease

Type of cut with COMPLEMENTARY OVERHANGS

Sticky end restriction endonuclease

Identify the type of location of cut:

• Makes a cut randomly as far as 1,000 bps away from the recognition site.

Type I

Identify the type of location of cut:

• makes a cut directly within the recognition site

Type II

Identify the type of location of cut:

• makes a cut usually within 25 bps of the recognition site

Type III

RFLP stands for _

Restriction Fragment Length polymorphism

This is a technique that uses variations in homologous DNA sequences to distinguish between individuals, determine ancestry and family relations, identify spp., or pinpoint the location of genes within a sequence

RFLP

Type of blotting used in RFLP

Southern Blot

The process of characterizing an unknown segment of DNA through restriction enzyme

digestion and then mapping the location of the restriction sites through electrophoresis.

Restriction enzyme mapping

TRUE OR FALSE: Probes are always labelled

FALSE

Solid support hybridization is AKA

Blotting

Identify the solid support hybridization technology:

• samples applied to a membrane, heated to denature, labelled, then washed

Dot/Slot blot

Identify the solid support hybridization technology:

• ONE SAMPLE can be tested but a lot of genetic markers to find

Reverse dot blot

Identify the solid support hybridization technology:

• utilizes 2 probes: capture and reporter probe

Sandwich hybridization

TRUE OR FALSE: Nucleic acid strands must first be denatured because probes will not anneal to double-stranded DNA or RNA

TRUE

This refers to the temperature at which 50% of a given sequence is double stranded; other sequence is single stranded na

Melting temperature

Ideal hybridization/annealing temperature will be found approx. how many degrees lower than the melting temperature

3-6 C lower than Tm

Relationship of probe length and time to hybridize

Direct