Download ƒ Announcements  ƒ Pre‐lab Lecture ™ Module 2: Design Overview ™ Primer design for mutagenesis

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Transcript
ƒ Announcements ƒ Pre‐lab Lecture
™ Module 2: Design Overview
™ Primer design for mutagenesis
™ Intro to Restriction Enzymes
™ Today in Lab: M2D1
Announcements
• iGEM competition
• Introducing… Xiaosai, your TA for Module 2
Inverse Pericam
EYFP
145
238 1
144
cp EYFP
calmodulin CaM
Æbinds M13 if Ca2+ bound
Æ4 Ca2+ binding sites
Courtesy of National Academy of Sciences, U. S. A. Used with permission.
Source: Nagai, T., et al. “Circularly Permuted Green Fluorescent Proteins
Engineered to Sense Ca2+.” PNAS 98, no. 6 (March 6, 2001): 3197-3202.
Copyright © 2001 National Academy of Sciences, U.S.A.
Goal: Affect Binding Properties
Designing Mutagenic Primers
5’
TCGAT
/ CTA
AG
T
A
TC
/ GAT
Intro to Restriction Enzymes
G A A T T C
endonucleases
Æcut DNA
C T T A A G
TGC GCA
ACA CGT
“sticky ends” can be used in ligations
G AATTC
CTTAA G
palindromic DNA
also type 2
leave blunt ends
Today in Lab
• Study inverse pericam at multiple levels • Design primers
silent mutation
– Amino acid change
– Silent change make new, unique restriction site
• For next time: begin reading two papers
– Focus on Nagai; other one is for some history/context
– Time in class on D2 to finish
MIT OpenCourseWare
http://ocw.mit.edu
20.109 Laboratory Fundamentals in Biological Engineering
Spring 2010
For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.