Biochemistry 2280A Lecture Notes - Stop Codon, Tryptophan, Methionine

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An absolute minimum of 1000 clones would be needed because to be sure you have full coverage you want a library with an excess of several fold (about 20). Creating a genetic dna library for h. influenza: Starting with millions of cells, you extract the dna from h. influenza and sonicate it (instead of restriction enzymes) into random dna fragments of various sizes. You then purify the fragments through gel electrophoresis and select for those fragments around 2000 base pairs in size. By cloning these 2000 base pair fragments, you receive 20,000 clones (20-fold excess), where each clones represents and independent fragments of the genome. Next, you isolate the plasmids from the 20,000 different clones and sequence them through dideoxynucleotide sequencing. The result is 25,000 sequences from 20,000 clones and approximately 12 million base pairs of sequence altogether (lots of redundancy present).

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