Cloning 1 Flashcards
(19 cards)
Tm is determined by their ____ and _____
size and GC content
How can you increase TM
increase GC content
Increase the length
Primers always become part of the _____
product
Discuss the considerations for primers B and C
adeoquate verlap for efficient splicing
Will contain the mutation at the site of interest and surrounding sequences
18-35 nt in length
A and D primers
Determine the size of the final product
Can be used to introduce recognition sites for enzymes
What are considerations for choosing a primer?
Both forward primers will be the same sequence
Recombinant DNA
any DNA molecule that is made of a segment or fragment of another source
DNA cloning
preparing large numbers of identical DNA molecules
Why clone DNA?
To study their structure, function, and how they are regulated
Basic scheme of cloning
- Vector and DNA fragment
- Recomb DNA
- Rep of Recomb DNA within host cells
- Isolation, seq and manipulation of purified DNA fragment
Principles of modification
Bacterial DNA is methylated
Methyl groups protect against attack from restriction enzymes
Methylation patterns are maintained during DNA replication
What happends to DNA that is unmethylatedd?
It is degraded
Restriction enzymes were named because
They restricted or prevented viral infection by degrading invading DNA
Types I and III
Are not very useful because they cleave DNA at sites away from the recognition site and produce random cleavage patterns
Type II
recognize specific sites and cleave at these sites
Type II restriction enzymes
-Recognize pallindromic DNA seq 4-8bp
-sticky ends
-blunt ends
DNA ligase catalyzes formation of ____ betwenn __ and ___
phosphodiester bond between 5’ phosphate and 3’ hydroxyl
DNA ligase requires
ATP co factor
