User:Isis Trenchard/Notebook/BioE44 Stuff/2010/03/24: Difference between revisions

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''Version 2''
''Version 2''
'''Day 2'''
 
'''Day 2 (1)'''
*give them cultures with color generator vectors under inactive promotor? so no color expression
*give them cultures with color generator vectors under inactive promotor? so no color expression
*miniprep
*miniprep
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*take digests, heat inactivate and store
*take digests, heat inactivate and store


'''Day 3'''
'''Day 3 (2)'''
*make gel
*make gel
*load digest
*load digest
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*put away ligations
*put away ligations


'''Day 4'''
'''Day 4 (3)'''
*electrocompetent cell prep
*electrocompetent cell prep
*transformation
*transformation
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*pick colonies
*pick colonies
*set up cultures with different concentrations of inducer (have them calculate stuff ahead of time)
*set up cultures with different concentrations of inducer (have them calculate stuff ahead of time)
'''Day 5 (4)'''
*spin down cells
*extract pigments
*look at full absorbance spectrum
*data analysis


==Other stuff left to order==
==Other stuff left to order==

Revision as of 14:36, 24 March 2010

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Reworking the first part of the class

  • realized that the way the first two weeks are planned right now, can't get everything done.
  • how to rework so students still learn cool stuff but will feasibly get things done?
  • need to order arsenic sensor
  • want students to think thru things and make decisions - not too hand-holdy

Version 2

Day 2 (1)

  • give them cultures with color generator vectors under inactive promotor? so no color expression
  • miniprep
  • nanodrop
  • digest

Day 2.5 (TAs)

  • take digests, heat inactivate and store

Day 3 (2)

  • make gel
  • load digest
  • run gel
  • cut bands, extract dna from gel
  • set up ligation (vectors already prepped by TAs - can choose type of inducible?)

Day 3.5 (TAs)

  • put away ligations

Day 4 (3)

  • electrocompetent cell prep
  • transformation
  • plate

Day 4.5

  • pick colonies
  • set up cultures with different concentrations of inducer (have them calculate stuff ahead of time)

Day 5 (4)

  • spin down cells
  • extract pigments
  • look at full absorbance spectrum
  • data analysis

Other stuff left to order

  • tubing for bunsen burners and aspirators
  • cellophane discs for moss culture
  • dissecting microscope?
  • inverted microscope?
  • acetone

Pigment extraction protocol

  • from cambridge 2009
  • We grew transformed E.coli MG1655 in LB culture at 37°C for 24 hours and collected cell pellet by centrifugation. Acetone was added to the cell pellet and warmed at 50°C for 10 minutes, allowing carotenoids (lycopene or β-carotene) in the cells to dissolve. The acetone extracts were then put into MicroPlate Reader for a full photospectrum scan (wavelengths between 300nm and 800nm). The absorbance curves showed peaks characteristics of the respective carotenoids: at wavelength 474nm for lycopene, and 456nm for β-carotene. I


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