A radioactive probe can take what forms?
Probes can be
Name the two ways probes can be designed
What is codon bias?
Where cells prefer one codon of an aa to another
Where cells will reject certain codons due to what they contain
Where some cells can only make tRNA's for certain codons
80% of Phe codons are TTC, 20% are TTT
If the protein is an enzyme it can be found by adding the substrate
State the possible forms of Ig's for protein separation?
Immobalised on a column
Removable from solution
Probes needn't be 100% accurate to work
For a Western Blot, how is the DNA stuck to the membrane?
Denatured DNA sticks to membrane
Bacteria are lysed to release DNA
DNA washes off
Once the DNA has been bound, what happens next?
A probe is added
The membrane is run through a gel
The membrane is stained
In an expression vector, where is the new gene added?
The Multiple Cloning Site
The Promotor Region
Next to the origin
Name the parts on the vector
Multiple Cloning Site
More compliated vectors have proteins tagged for purification
What is involved in His Chain Purification?
The addition of a His chain to the N terminus
The removal of a His chain from the protein
The use of a His chain to cut unwatned proteins
His Purification uses Histadine's affinity for what metal?
His-tagged proteins will bind to Nickel in a column. Others that don't bind will be washed off.
The PET system involves using a promoter for the protein that you can control the expression of
What promoter is used in the PET system to control protein expression?
In the PET system, what promoter is used to control the T_ expression?
What is required for a good shuttle vector?
Replicable in both hosts
Selectable in both hosts
Expressable in both hosts
Shuttle vectors are usually plasmids with additional sequences
Shuttle vectors for yeast/fungi need
A baclovirus would be used as a vector into what?
Secretion of certain proteins can be caused by what?
Why are there two origins in the Yeast shuttle vector?
One for the yeast to use
One for the bacteria to use
What is the method of selection in yeast?
URA3- allows growth in the absence of Uracil
TRA3- allows growth in the absence of thymidine
GRA3- allows growth in the absence of guanine
Like bacteria, yeast cannot take up linear DNA
What does a YAC contain that a plasmid does not?
Yeast can allow DNA to be directly inserted into its genome
How can yeast accept DNA into its genome?
Non-homologous end joining
In order to directly insert a gene, you must flank the gene with identical stretches of the part of the genome you want to insert it in
Plants would use an aglobacterium T1 plasmid
How do you replicate transformed plant cells?
Grown them as plantlets
Asexually reproduce them
Mammal shuttle vectors are the same as yeast vectors, but the yeast origin is replaced with what?
An SV40 promoter
A UG35 promoter
A FT12 promoter
Retroviruses can also be used to insert transgenes into mammilian genomes by packing the transgene in a retroviral capsule