Class Notes (859,559)
CA (520,842)
Western (50,903)
Biochemistry (856)
2280A (644)
Eric Ball (118)
Lecture 9

# Brandl Lecture 9

3 Pages
186 Views

Department
Biochemistry
Course Code
Biochemistry 2280A
Professor
Eric Ball

This preview shows page 1. Sign up to view the full 3 pages of the document.
Description
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
More Less
Subscribers Only

Only page 1 are available for preview. Some parts have been intentionally blurred.

Unlock Document
Subscribers Only

Unlock to view full version

Unlock Document
Subscribers Only
Notes
Practice
Earn
Me

OR

Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Join to view

OR

By registering, I agree to the Terms and Privacy Policies
Just a few more details

So we can recommend you notes for your school.