MIMG 101 Lecture Notes - Lecture 15: Shotgun Sequencing, Replica Plating, Gene Gun

• School: UCLA
• Department: Microbiology, Immunology, and Molecular Genetics
• Course: MIMG 101
• Professor: G U N S A L U S& B R A D L E Y

Document Summary

In vitro adaptor ligation added (know adaptor sequences) Generate polony array (pcr colony make more copies of fragments) Sequence thru cyclic array sequencing: works one base at time. Get rid of fluorescence and unblock by reversing: generate short reads thru different cycles. 36 bp per go but can do many reactions at a time (= 36 million bp) still shotgun sequencing, still has overlaps: needs reference strain to figure out overlaps. Not affected by toxic genes like shotgun sequencing, but repeats are still problem can do genome resequencing, large scale polymorphisms, look at microbiome, can sequence mutants, study metagenomics shotgun sequencing. In vivo cloning into plasmid and amplify in bacteria on plate to purify. Gives 800 1000 bp per run est library = sequence of reverse library of cdna from mrna. # reads = # mrna = # cdna. Better at looking at rna quantities still need reference strain bacterial uptake of dna.