Plasmids Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates (has an Ori ) inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) 10 -100 copies per cell called high copy number 1- 4 copies, low copy number
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Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates (has an Ori ) inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb)
10-100 copies per cell called high copy number
1-4 copies, low copy number
Many exist naturally in
Many recombinant forms
have been designed for
use in cloning
Different kinds of plasmids that exist in nature
F plasmids (fertility)
2. pUC family of vectors
What are the ideal features of a cloning vector such as a plasmid?
Steps in cloning a piece of DNA
Making a Recombinant plasmid
1. Sticky ends
2. poly tailing technique
3. Blunt end ligation
4. Chemicallly synthesized linkers can be made. Disadvantage is still have to blunt end to stick them on, advantage is can insert a Restriction site and can recover insert easily.
e.g. Streptococcus pneumonia (gm +ve)