BACs

Question 1

What is recombinering?

Answer 1

Once specific DNA cloned into BAC, a GFP sequence can be recombinered. 
Cholera toxin (cell death)
rhodopsin channel (neural activity inhibition) 
can also be added. 

Recombinered molecules expressed in same way as specific DNA (gene X).

Question 2

What is GENSAT?

Answer 2

NOH publicly funded gene expression at.as of developing and adult CNS in mice.

BAC-EGFP reporter and BAC-Cre recombinase dimer mouse lines available as research tools.

Question 3

What is a BAC library?

Answer 3

Store of genomic DNA fragments.

DNA isolated, fragmented and separated by electrophoresis.
Fragments LIGATED into dephosphorylated BAC vectors.
BACs TRANSFORMED into E.Coli via electroporation.
E.Coli grown and stored.

Question 4

what ar the goals of the yeast 2 hybrid system?

Answer 4

Confirm interaction between two known proteins.

Identify ‘new’ interacting proteins.
known = bait
interacting = prey.

Question 5

What is a replica filter?

Answer 5

Bacteria attached to a membrane.

Question 6

Outline how you would clone a gene in order to produce a certain quantity of its product?

Answer 6

Isolate the cDNA of the gene

Clone into vector

Make cell lines producing protein

Harvest cells and extract protein.

Question 7

How do you know what probe to design?

Answer 7

Isolate protein

Determine partial sequence

Calculate gene sequence

Design probe.

Question 8

What is a BAC?

Answer 8

Plasmids based on e.coli F plasmid

Can accommodate large inserts up to 300kB

These can be transformed into E.Coli