20.109(F12):Module 1

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (09:48, 11 September 2012) (view source)
(Module 1)
 
(4 intermediate revisions not shown.)
Line 1: Line 1:
-
[[https://20109blog.21classes.com/home/registerUser1.htm?slsid=9392982807176794]]
+
{{Template:20.109(F12)}}
 +
 
 +
<div style="padding: 10px; width: 640px; border: 5px solid #009966;">
 +
 
 +
==Module 1==
 +
 
 +
'''Instructors:''' [http://web.mit.edu/be/people/engelward.htm Bevin Engelward], [[User:Shannon K. Alford |Shannon Hughes-Alford]], [[Natalie Kuldell]], [[User:AgiStachowiak| Agi Stachowiak]]
 +
 
 +
'''TA:''' Jenny Kay (Engelward Lab) <br>
 +
 
 +
In this experimental module you will modify the gene for ''EGFP'' (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event.  We will then choose a few variables to test on the final day of the experiment.
 +
 
 +
[[Image:Be109recombomouse.jpg|thumb|left|200px|'''Recombocell image from Dominika Wiktor of the Engelward Lab''']]
 +
[[Image:Experimental Overview.jpg|thumb|right|350px|'''A schematic overview of the module.''']]
 +
<br style="clear:both" />
 +
 
 +
==Lablinks: day by day==
 +
[[20.109(F12): Mod 1 Day 1 DNA engineering using PCR| Day 1: DNA engineering using PCR]]<br>
 +
[[20.109(F12): Mod 1 Day 2 Clean and cut DNA | Day 2: Clean and cut DNA]]<br>
 +
[[20.109(F12): Mod 1 Day 3 Agarose gel electrophoresis| Day 3: Agarose gel electrophoresis]]<br>
 +
[[20.109(F12): Mod 1 Day 4 DNA ligations and bacterial transformations| Day 4: DNA ligation and bacterial transformation]]<br>
 +
[[20.109(F12): Mod 1 Day 5 Examine candidate clones & tissue culture| Day 5: Examine candidate clones & introduction to tissue culture]]<br>
 +
[[20.109(F12): Mod 1 Day 6 Lipofection & lab practical| Day 6: Lipofection & lab certification]]<br>
 +
[[20.109(F12): Mod 1 Day 7 FACS analysis| Day 7: FACS analysis]]<br>
 +
 
 +
==Assignments==
 +
[[20.109(F12): Module 1 celebrations of learning| summary of Module 1 assignments]]<br>
 +
[[20.109(F12): Lab certifications| DNA engineering lab certifications]]<br>
 +
[[20.109(F12): Online cloning lab and defense| Online cloning lab and defense]]<br>
 +
[[20.109(F12): FACS data analysis and defense| FACS data analysis and defense]]<br>
 +
[[20.109(F12): M1 ppt summary and notes| DNA engineering powerpoint summary and notes]]<br>
 +
[[20.109(F12): Reflections| blog assignment]]
 +
 
 +
==References==
 +
#'''DNA double-strand break repair: From mechanistic understanding to cancer treatment'''<br>''DNA Repair'' 2007<br> Thomas Helleday, Justin Lo, Dik C. van Gent, Bevin P. Engelward<br> [http://web.mit.edu/engelward-lab/publications.htm URL] <br>[http://web.mit.edu/engelward-lab/animations/DSBR.html Sample Animation] <font color = 9900CC>Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!</font color><br>
 +
#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://dx.doi.org/10.1016/S0304-419X(01)00018-X URL]
 +
#'''Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death'''<br>'' EMBO J'' 15 January 1998<br> E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda M <br> [http://www.nature.com/emboj/journal/v17/n2/abs/7590776a.html URL]
 +
 
 +
==Notes for Teaching Faculty==
 +
[[20.109(F12): TA notes for module 1| TA notes, mod 1]]
 +
[[20.109(S12): TA notes for orientation| S12 notes for orientation day]]

Current revision

20.109(F12): Laboratory Fundamentals of Biological Engineering

Home        People        Schedule Fall 2012        Assignments        Lab Basics        OWW Basics       
DNA Engineering        System Engineering        Biomaterials Engineering              

Contents

Module 1

Instructors: Bevin Engelward, Shannon Hughes-Alford, Natalie Kuldell, Agi Stachowiak

TA: Jenny Kay (Engelward Lab)

In this experimental module you will modify the gene for EGFP (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment.

Recombocell image from Dominika Wiktor of the Engelward Lab
Recombocell image from Dominika Wiktor of the Engelward Lab
A schematic overview of the module.
A schematic overview of the module.


Lablinks: day by day

Day 1: DNA engineering using PCR
Day 2: Clean and cut DNA
Day 3: Agarose gel electrophoresis
Day 4: DNA ligation and bacterial transformation
Day 5: Examine candidate clones & introduction to tissue culture
Day 6: Lipofection & lab certification
Day 7: FACS analysis

Assignments

summary of Module 1 assignments
DNA engineering lab certifications
Online cloning lab and defense
FACS data analysis and defense
DNA engineering powerpoint summary and notes
blog assignment

References

  1. DNA double-strand break repair: From mechanistic understanding to cancer treatment
    DNA Repair 2007
    Thomas Helleday, Justin Lo, Dik C. van Gent, Bevin P. Engelward
    URL
    Sample Animation Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!
  2. Homologous recombination as a mechanism of carcinogenesis
    Biochim Biophys Acta 21 March 2001
    Bishop AJ and Schiestl RH
    URL
  3. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
    EMBO J 15 January 1998
    E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda M
    URL

Notes for Teaching Faculty

TA notes, mod 1

S12 notes for orientation day
Personal tools