Gene cloning and manipulation
6 important questions on Gene cloning and manipulation
How do we isolate and manipulate gene fragments?
What are restrictions enzymes?
- Type II: one enzymes recognizes and cuts the actual sequence and another enzyme methylates
- recognize palindromes
- works as dimers to recognize the sequence in both strands
- create sticky or blunt ends when cutting DNA
What are the difference between sticky and blunt ends?
- the vector needs also te be sticky so it can bind to the plasmid very effective
- blunt ends are not limited like sticky, but are ineffective
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What is pasting (ligating)?
What are the specifications of a cloning vector?
- Origin of replication
- high copy number
- selection marker (antibiotic resistance gene)
- They help in identifying transformants and selectively allowing their growth whereas eliminating non-transformants in cloning vector
- unique restriction sites to inset DNA
- simple method to establish recombinants (blue/white screening)
Why are vectors used with topoisomerase?
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