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

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==Pigment extraction protocol==
==Pigment extraction protocol==
*from cambridge 2009
*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
*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.
 
==Biobrick plasmids and promotors==
*LacI promotor (BBa_R0011) - inducible by IPTG
** 2009 distribution - Kit plate 1 - 6G - pSB1A2
*pBAD promotor (BBa_I0500) - inducible by arabinose
** 2009 distribution - kit plate 1 - 6G - pSB2K3


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Revision as of 16:45, 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 (Thurs April 1)

  • give them cultures with color generator vectors under inactive promotor? so no color expression under constituative promotor so they can see colors and start thinking about how to measure input/output, understanding behavior etc. Also read about and understand how their color generators work - give them parts numbers so they can explore the registry?
  • miniprep
  • nanodrop
  • digest

Day 2.5 (Fri April 2, TAs)

  • take digests, heat inactivate and store

Day 3 (Tues April 6)

  • 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? either arabinose or iptg? high copy/low copy...)

Day 3.5 (Wed April 7, TAs)

  • put away ligations
  • TA's set up cultures for electrocomp. prep

Day 4 (Thurs April 8)

  • electrocompetent cell prep
  • transformation
  • plate

Day 4.5 (Fri April 9)

  • TA's put plates away

Day 4.5 (Mon April 12 - students)

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

Day 5 (Tues April 13)

  • 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.

Biobrick plasmids and promotors

  • LacI promotor (BBa_R0011) - inducible by IPTG
    • 2009 distribution - Kit plate 1 - 6G - pSB1A2
  • pBAD promotor (BBa_I0500) - inducible by arabinose
    • 2009 distribution - kit plate 1 - 6G - pSB2K3


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