Lab Techniques

Front 1

I want to amplify a desired fragment of DNA

What technique and what are the steps

Back 1

PCR

1. denature DNA by heating --> 2 separate strands

2. Annealing strands during cooling by providing excess DNA primers to anneal to each strand

3. Elongation by heat-stable DNA polymerase, which adds bases following each primer

Repeat

Front 2

After PCR, why do agarose gel electrophoresis?

Back 2

to size separate PCR products or compare against a DNA ladder

Front 3

What molecules does each involve
a. southern blot
b. northern blot
c. western blot

Back 3

a. DNA
b. RNA
c. protein

Front 4

A sample is electrophoresed on a gel, transferred to a filter --> filter exposed to denaturant and radiolabeled probe that binds and allows for visualization of sample when filter is exposed to film

a. what process?
b. what if the sample is DNA?
c. What if the sample is RNA?
d. What if the sample is protein

Back 4

blotting
a. southern
b. northern
c. western

Front 5

I want to profile gene expression levels for thousands of genes

what should I do?

Back 5

Microarray

Arrange nucleic acid sequences in grids on glass or silicon, hybridize DNA/RNA probes to the chip, use a scanner to detect relative amounts of complementary binding

Front 6

How sensitive is a microarray for detecting gene expression

what are some areas of application of this

Back 6

can detect single nucleotide polymorphisms

Used in genotyping, forensics, predisposition to disease, cancer mutations, genetic linkage analysis

Front 7

I want to test antigen-antibody reactivity

What test and how

Back 7

Enzyme-linked immunosorbent assay (ELISA)

probe patient's blood with radiolabeled
a. antigen to see if antibody is present
b. antibody to see if antigen is present

Produces color

Front 8

I want to localize a gene or find a genetic microdeletion that is too small to be seen with karyotyping

what should I do

Back 8

FISH

Fluorescent DNA/RNA probe binds to gene site of interest

Front 9

I want to produce recombinant DNA that is self perpetuating

method

Back 9

Cloning

Isolate tissue mRNA, expose to reverse transcriptase --> cDNA librabry (no introns)

expose DNA to restriction enzymes that cleave DNA at 4-6bp palindromic sequences --> insertion into bacterial plasmids that contain antibiotic resistence genes

select for plasmid-containing bacteria by using antibiotic plates

Front 10

What is the difference between constitutive and conditional gene expression modification studies?

Back 10

constitutive = random insertion of gene into mouse genome

conditional = targeted insertion or deletion of gene through homologous recombination with mouse gene

Front 11

I want to inducibly manipulate genes at specific developmental points

ex: i want to study a gene whose deletion causes embryonic death

I have an antibiotic controlled promoter

What process should I undergo

Back 11

Cre-lox to study deletions

Cre = DNA recombinase, cleaves DNA at lox P sites, rejoined by ligase

Front 12

What is the RNAi system?

Back 12

Used to knockout genes

dsRNA synthesized that is complementary to mRNA of interest --> broken down to ssRNA in human body--> binds to and promotes degradation of target mRNA

Front 13

I want to diagnose chromosomal imbalances (autosomal trisomies, sex chromosome disorder)

what process
where can i get samples
how do i organize my results

Back 13

karyotyping

get a sample of blood, bone marrow, amniotic fluid, or placental tissue

Take metaphase chromosomes --> stain, order, and number according size, morphology, arm-length ratio, banding pattern