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Lecture 9

Brandl Lecture 9

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Biochemistry 2280A
Eric Ball

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Biochem Lecture 9 - Nov. 25th Case Study (con’t) Restriction Enzyme Cleavage • varies for different enzymes • different enzymes cleave different sequences and leave different end styles • Cleavage by EcoRI - leaves “sticky ends” with 5’ overhangs • Cleavage by Kpnl - leaves sticky ends with 3’ overhangs • Cleavage by Sspl - leaves blunt ends • cleaves down the middle • some restriction enzymes cut identical sequences but leave different overhanging ends • some restriction enzymes cut different recognition sequences but leave identical ends (ex. BamHI and BgIII) • important because similar overhangs can be ligated together • Question: What is the probability that any random 4 bp sequences is a Haelll site (GGCC) • Answer: 1/256 • probability that the 1st position is a G is 1 in 4 • probability that the 2nd position is a G is 1 in 4 • 1 in 4 for 3rd position • 1 in 4 for 4th position • therefore 1 in 4 times 4 position = 1 in 256 • Question: Given that the probability of any 6bp sequence being an Hincll site (GTPyPuAC) is approx. 1/1000, approximately how many Hincll sites are dounf within the human genome (3 billion bp) • Answer: About 3 million • 3x10^9/10^3 = 3x10^6 Restriction Enzymes are Enzymes • restriction enzymes have ideal conditions just like any other enzymes • temp • pH • salt concentration Restriction Enzymes have reaction kinetics • over time, % DNA digested increases fairly linearly DNA Ligase: • DNA ligase = glue for DNA • will reseal compatible sticky ends, and blunt ends (much less efficiently) • requires and energy source (ATP) • need the phosphates on the 5’ ends of the DNA in order for ligase to work Step in Cloning: ← 1. digest approx. 100ng of YFG and a plasmid vector w
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