20.109(S15):Module 2: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 24: Line 24:
===Lab Links===
===Lab Links===


[[20.109(S15):Introduction to cell strains and plating (Day1) | Module 2 Day 1: Introduction to cell strains and plating]]<br>
[[20.109(S15):Introduction to cell strains and plating (Day1) | Day 1: Introduction to cell strains and plating]]<br>
[[20.109(S15):Begin Western protein analysis and choose system conditions (Day2) | Module 2 Day 2: Begin Western protein analysis and choose system conditions]]<br>
[[20.109(S15):Begin Western protein analysis and choose system conditions (Day2) | Day 2: Begin Western protein analysis and choose system conditions]]<br>
[[20.109(S15): Complete Western and prepare damaged DNA (Day3)| Module 2 Day 3: Complete Western and prepare damaged DNA]]<br>
[[20.109(S15): Complete Western and prepare damaged DNA (Day3)| Day 3: Complete Western and prepare damaged DNA]]<br>




Line 32: Line 32:




[[20.109(S15):Cell preparation for DNA repair system (Day4)| Module 2 Day 4: Cell preparation for DNA repair system]]<br>
[[20.109(S15):Cell preparation for DNA repair system (Day4)| Day 4: Cell preparation for DNA repair system]]<br>
[[20.109(S15):DNA repair assays (Day5)| Module 2 Day 5: DNA repair assays]]<br>
[[20.109(S15):DNA repair assays (Day5)| Day 5: DNA repair assays]]<br>
[[20.109(S15):Data analysis and paper discussion (Day6)| Module 2 Day 6: Data analysis and paper discussion]]<br>
[[20.109(S15):Data analysis and paper discussion (Day6)| Day 6: Data analysis and paper discussion]]<br>
[[20.109(S15):]]<br>
[[20.109(S15):]]<br>



Revision as of 13:21, 15 December 2014


20.109(S15): Laboratory Fundamentals of Biological Engineering

Home        People        Schedule Spring 2015        Assignments        Lab Basics        OWW Basics       
DNA Engineering        System Engineering        Biomaterials Engineering              

Module 2: System Engineering and Protein Foundations

Lecturer: Leona Samson
Instructors: Shannon Hughes, Noreen Lyell, and Leslie

TA: [[User:]]

Overview

During the first module, you spent part of your time designing and testing a diagnostic tool for detecting a microorganism. Now you will have the opportunity to apply a research tool that detects a molecular process to address an interesting biological question. Specifically, you will investigate the importance of different proteins to a type of DNA repair called non-homologous end-joining, or NHEJ. Measuring DNA repair accurately and quantitatively can pave the way for certain cancer diagnostics and therapeutics.

This time the DNA engineering has been done for you: the plasmid-based research tool to measure NHEJ already exists, along with a novel version developed just for 20.109. In this second module you will instead take a systems level view, as you explore how different topologies of DNA damage are repaired by wild-type hamster (CHO) cells, CHO cells treated with a small molecule inhibitor of an NHEJ protein, and CHO cells that are natively deficient in an NHEJ protein. Along the way, you will gain additional skills in analyzing and communicating information related not only to DNA assays (as in Module 1), but also to protein and cell-level assays. The culminating experiment will utilize flow cytometry, an amazing and infinitely useful technique that measures the fluorescence of individual cells. To evaluate class-wide trends in the flow data, you will learn and use basic statistical tools. The toolkit you develop during the first two modules should leave you well poised to tackle the third and final module.

We thank Zac Nagel from Samson lab for his many contributions to the development of this module, along with Carrie Thompson for assistance with irradiation assays. We also gratefully acknowledge Integrated DNA Technologies for speedy assistance with cloning/expression troubles that we encountered during module pilots.

Module 2 Sample Data. The two primary techniques that you will learn to carry out and interpret in this module are flow cytometry (left) and Western blotting (right). We will use flow cytometry to quantify DNA repair events, in concert with a two color co-transfection assay. We will use Western blotting to evaluate DNA repair protein levels; the sample data shows that one of the cell lines is deficient in a particular NHEJ repair protein.


Lab Links

Day 1: Introduction to cell strains and plating
Day 2: Begin Western protein analysis and choose system conditions
Day 3: Complete Western and prepare damaged DNA


Note: spring break occurs between Days 3 and 4.


Day 4: Cell preparation for DNA repair system
Day 5: DNA repair assays
Day 6: Data analysis and paper discussion
20.109(S15):

TA notes, mod 2

Assignments

NHEJ system research article