Class Notes (836,865)
Canada (509,936)
Biochemistry (847)
Eric Ball (118)
Lecture

Biochem Brandl 7

4 Pages
140 Views
Unlock Document

Department
Biochemistry
Course
Biochemistry 2280A
Professor
Eric Ball
Semester
Fall

Description
Biochem - Lecture 7 - Nov. 22th Case Study: • You have identified a human protein that will be of tremendous value in treating disease. Your goal is to mass produce this protein (YFP) Expression of YFP using RDT: • many proteins are difficult to obtain from their native source • RDT can dramatically increase expression and facilitate purification • E. coli are the most commonly used organism for expressing YFP (true for almost all protein - always the one you try first) • grow quickly and inexpensively • protein extracts are made easily • genetic engineering is simple and done for decades • multi-copy plasmids and strong promotors can drive expression Obtaining YFP will require: ← 1. cloning Your Favorite Gene 2. introducing YFG into E. coli 3. purifying the protein from E. coli How do we get enough of YFG to be able to clone it? • synthetic oligonucleotides • hybridization • PCR Synthetic Oligonucleotides: • short fragments (20-100 bases) of single stranded DNA will defined sequence can be made synthetically and inexpensively • often referred to as primers or oligos Nucleic Acid Hybridization: • denaturation/melting • renaturation/annealing/hybridization • DNA denaturation: • if you heat a double strand DNA or put it into a high pH, the strands will separate into single stands Melting Temperature: • given a specific DNA at a concentration of 1 M, the temperature at which 50% of the molecules are single stranded (referred to as the “Tm”) • every different DNA molecule has a different TM Factors that determine the Tm: • intrinsic features of the molecules itself • PolyG/C’s have a higher Tm than PolyA/T’s • length of the strand - longer the DNAs have higher a Tm then shorter molecules (due to more interactions) • Tm = 4 degrees (G:C) + 2 degrees (A:T) • degree of complementarity between the two nucleic
More Less

Related notes for Biochemistry 2280A

Log In


OR

Join OneClass

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

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit