Outline how positive and negative selection are used to obtain targetted knockouts in mouse embryonic stem cells.

Why do we need both positive AND negative selection?

Provide a rationale for why injection of ES cells into the 8 cell embryo result in mice derived entirely from the injected cells when injection into the blastocyst produces mosiac mice made up of both injected cells and blastocyst cells?

What are cre/lox and flp/frt.

Describe the results of cre mediated recombinantion between two homotypic loxP sites
when they are in cis and in the same orientation.
when they are in cis and in the opposite orientation
when they are in trans on homologous chromosomes
when they are in trans on non-homologous chromosomes

In order to examine the function of large genomic regions that lack protein coding potential
and yet show evolutionary conservation, site specific recombination was used as a tool to create defined deletions and duplications of the conserved regions.
Diagram the arrangements of loxP sites that can be used for such experiments.

How are we able to use site specific recombination to engineer precise replacements of mouse genomic sequences with human syntenic regions?

Explain why the formation of DNA duplex (reassociation) is a concentration depentant phenomenon.

Draw the Cot curve illustrating the reassociation of genomic DNA from an E. coli lambda lysogen.
Explain the shape of the curve you have drawn.

Draw the Cot curves for reassociation of human genomic DNA.
Explain the shape of the curve.

Draw the Cot curve for the reassociation of a randomly primed cDNA population.
Explain the shape of the curve.

On the above curves, show the reassociation of the radioactive probes:
actin cDNA
histone cDNA
TATA-Binding Protein cDNA

Explain the term NORMALIZE.
How does reassocation kinetics enable you to normalize a cDNA library?

Explain the significance of salt concentration when manipulation hybridization stringency.

The human genome is 3,000,000,000 bp long and 50% GC
You cut 5 ug of human genomic DNA with the enzymes BamHI, EcoRI, HindIII and XbaI
(all are 6-mers)
You size fractionate the DNA on an agarose gel
Draw a picture showing the ethidium bromide stained pattern of fragments.
You denature the DNA and transfer it to a nylon filter.
You label a cDNA probe and hybridize in 0.1M Na+
What temperature do you hybridize at? (calculate)
You wash off unbound probe under the same conditions.
How much mismatch do these conditions tolerate?
You repeat the wash at 65 deg C and 0.05 M salt.
How much mismatch do these conditions tolerate?
You repeat the was at 65 deg C and 0.01M salt.
How much mismatch do these conditions tolerate?

Outline the modifications made to the primary transcript in eukaryotes.

Draw the structure of the mRNA's 5' CAP.

Outline how introns are removed from the primary transcript and how the exons are joined.

How is the 3' end of the mRNA generated?

How are these modifications coordinated in the eukaryotic nucleus?

What is nonsense surveillence?
How does it work?