plasmid dna isolation and electrophoresis
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bacteria's circular chromosome contains genes necessary for:
- cell division
how many base pairs is in E. coli chromosome?
extra chromosomal circular DNA is called what and what is it used for?
- antibiotic resistance
how does cloning work?
- fragment of DNA is introduced to cell
- cell replicates
problem with cloning
lack of an origin of replication, cellular replication machinery will not copy
how to overcome cloning problem
- clone DNA linked to a cloning vector
- new cell DNA clones both of them
3 essential features of cloning plasmids
- origin of replication
- antibiotic(ampicillin) resistance gene
- cloning site
how fast do bacteria cells reproduce plasmid copies
10 million daughter cells overnight=20 billion copies of plasmid
what does the antibiotic resistance gene do?
allows growth in the presence of antibiotic
plasmid we are using today and what is it resistant too if no plasmid is contained?
what does a cloning site do?
- unique restriction enzyme recognition site
- when a vector is digested ww/restriction enzyme it opens up the plasmid
- all foreign DNA to be ligated within it
6. basic steps of plasmid insert
- 1. plasmid digested w/ restriction enzyme
- 2. opens plasmid
- 3. addition of forign DNA
- 4. ligases
- 5. plasmid w/insert
- 6. cloned DNA fragment
how will we isolate the plasmid(5 steps)
alkaline detergent solution, acidic salt, centrifuge, ethanol, centrigue
what does the alkaline detergent do?
- bind to protein in cell
- pH conditions will denature and shear
what's resistant to high pH conditions?
What does the acidic salt solution do?
- neutralizes the pH
- detergent precipitates
- forms matrix of broken DNA
whats in the pellets after the first centrifuge?
- denatured protein
- chromosomal fragments
What precipitates the plasmid DNA after the first centrifuge?
what are we determining with gel electrophoresis?
size of cloned insert
type of gel for gel electrophoresis
what charge is DNA and why
- phosphate backbone
which way will the DNA go?
migrates to the positive electrode(anode)
what size migrates faster?
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