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York University
BIOL 3110
Peter Cheung

Oct 6, 2009 Southern Blot Analysis -used in analyzing a digest fragment with a p probe, used for DNA fragments 9 -mammalian genomes are relatively large, with ~ 3 x 10 bp, the frequency of finding a desired site is dependent on how many bases the restriction enzyme requires (i.e. EcoR1 recognizes 6bps, therefore, (1/4) = 1/4096) -Once the fragments are processed in the gel it needs to be transferred onto a nitrocellulose filter and needs to be exposed via radiography. Northern Blot Analysis -Similar to the Southern Blot but this analyzes RNA -How to synthesize DNA: 5’ -----------------AAAAAA 3’ (3’OH)T T T T T T annealing olgio primer reverse transcriptase + 4 dNTP’s 5’ -----------------AAAA 3’ 3’ -----------------T T T T 5’ RNase H (digests RNA when RNA/DNA or alkaline) (3’OH) ---------------------------------5’ DNA polymerase + 4 dNTP + reverse transcriptase S1 nuclease (digests ss DNA or ss RNA cDNA probe with frayed ends -There are a few problems with these types of synthetic DNA: -Not full length cDNA -Both ends of the DNA probe are frayed -S1 nucleases will start to eat away the open ends -2* of DNA will halt reverse transcriptase Synthesizing Oligonucleotides (30 – 40 nt) -ca
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