Basic Cloning Methodologies Section II Flashcards
(16 cards)
Once bacteria are transformed with a plasmid what is necessary to do? And how can this be done?
It is necessary to screen for clones that contain the inserted DNA aka recombinants. This can be done by REPLICA PLATING.
How is replica plating performed?
Original cells are diluted and then the cells are plated on an agar plate. Wait. Colonies form. All colonies are derived fro one cell and are clones. Once colonies form take replica plating tool touch to cells and then place on new fresh plate in same pattern as the original plate = Replicate pattern colonies.
What other group of plasmid vectors were derived from pBR322?
pUC series of plasmids
What were the advantages of the pUC series of plasmids over the pBR322 plasmids?
- 40% of pBR322 DNA was removed
- Part of what was removed included the tetracycline resistance gene however the ampicillin gene remained intact
- Cloning sites in the pUC clustered into a small area called MCS (multiple cloning site)
- The MCS lies within a DNA sequence called lacZ’(italicized)
What antibiotic was pUC plasmids resistant to and sensitive to?
Resistant to ampicillin and sensitive to tetracycline
WHat does lacZ’ encode for?
the AMINO TERMINAL PORTION of beta-galactosidase
The host bacterium that uses pUC plasmids carries a gene fragment that codes for what?
The CARBOXYL PORTION of Beta-galactosidase
Can either the amino terminal portion of lacZ’ or the carboxyl portion of the host bacterium produce beta-galactosidase?
No
What is Alpha-complementation?
A process by which the two partial gene fragments, including the amino terminal portion of lacZ’ and the carboxyl portion of the host bacterium, cooperate together to form active beta-galactosidase.
What happens when pUC by itself without any inserted DNA is used to transform the appropriate bacteria?
And what happens when these bacteria are plated on an agar containing X-gal?
Beta-galactosidase is produced.
The normally colorless Xgal is cleaved by beta-galactosidase and the products of the reaction galactose and a colored molecule stain the bacteria blue.
What happens if the pUC plasmid is inserted with a gene and is used to transform bacteria?
ANd what happens when the bacteria is plated on an agar containing Xgal?
The lacZ’ in the pUC plasmid is disrupted and no amino terminal fragment is produced therefor no beta-galactosidase is produced.
The normally colorless Xgal remains colorless.
How can you screen for clones of plasmids?
Plating the bacteria on an agar plate that contains both ampicillin and Xgal. The colonies that grow and are white are the ones wanted and are picked and cultured.
Up to how many codons work in this system?
Up to 18 codons of foreign DNA work in this system. Anything larger and the integrity of the plasmid is destroyed
What is another advantage of plasmids with an MCS?
They allow for directional cloning.
How is directional cloning accomplished?
By cutting both the plasmid and the insert DNA with two different restriction enzymes. This ensures that the insert DNA can be placed into the vector in only one orientation with the ends of the insert matching their counterparts on the vector.
Directional cloning also prevents what?
the religation of the vector with itself
