Name the two methods of physical fragmentation
Shearing- filtering through a fine needed
Non-random shearing involves the use of helicases
Which of the following cuts most frequently?
A 4 cutter cuts a 4 bp sequence, and so on
The staggered ends of two different enzymes can be ligated together, but afterwards cannot be cut by the same enzymes
When dealing with DNA-cutting enzymes, what does the word degenerate mean?
The ends they produce don't last long
They can cut more than one sequence
Where in the sequence they cut changes
What conditions are necessary for a partial digest?
Not enough time
Wrong enzyme conditions
A four cutter
Why could a partial digest be done?
To get longer fragments and more likely preserve the gene
To save time
To prevent the DNA from degrading
Why is E.Coli most often used as a cloning vector?
It has a rapid generation time
Its DNA is easy to manipulate
Its DNA is easy to get hold of
They don't mutate much
Bacteriophages can be used to transplant vectors into E.Coli
How does M13 infect the male E.Coli?
Binds to pilus surface
Enters through bacterial pores
Direct injection of DNA
Once inside the cell, the ssDONA of M13...
Is convered to dsDNA that acts like a plasmid
Is integrated into the bacterial chromosome
Is cut up by restriction enzymes ans integrated into the bacterial chromosome
TEVEN and Lambda enter the bacteria directly through the pore
What is the point of the resistance genes in this Generation 1 plasmid
Tetracycline is interrupted- E.Coli with no resistance have had the recombinant plasmid inserted
The recombinant DNA is inserted at the EcrR1 site
E.Coli with ampicillin resistance have taken up a version of the plasmid
What gene is disrupted in Gen 2 plasmids?
The small subunit of LacZ
The large subunit of LacZ
Once the LacZ subunit has been assembled, what form does LacZ take?
For Gen 2 plasmid testing to work, the LacZ gene must be removed from the E.Coli, and a plasmid for the large LacZ subunit added
Why is the dish impregnated with X-Gal?
It goes from blue to white in the presence of LacZ
It goes from white to blue in the presence of LacZ
It goes from white to blue in the presence of the small subunit of LacZ
If the gene is successfully inserted, the colony will remain white
A Phagemid is virally inserted DNA in plasmid form
Why would a vector be given an M13 origin and inserted with M13?
The M13 phage will treat the plasmid as its own DNA and replicate it for secretion
The phage will insert the vector into the chromosome
The phage will prevent the bacteria from destroying the plasmid
Cosmids are plasmids with...?
Cos lambda sites
Cos beta sites
Cos gamma sites
Which of the following has the largest limit?
Why do you remove a phosphate from the ends of bacterial DNA using a phosphatase?
To prevent it self ligating
To prevent it degrading
To prevent it getting destroyed
Why mix enzyme T4 and ATP together with the DNA to be inserted?
Adding AMP to ligated ends gives easily ligable high energy form
ATP & T4 remove the phosphates from the ends of the DNA to prevent self annealing
T4 can only ligate DNA in the presence of ATP
A vector must be used because E.Coli cannot take up linear DNA