Davidson Protocols - Revision history

Macampbell at 21:24, 24 September 2019

2019-09-24T21:24:30Z

2019-01-13T19:15:21Z

2018-10-16T13:29:44Z

2018-10-16T13:28:20Z

2018-09-27T20:51:07Z

2018-09-27T20:49:27Z

2018-09-27T20:49:04Z

2018-08-28T21:06:58Z

2018-08-28T21:06:34Z

2018-08-27T20:34:39Z

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 '''I. Making Selective Media'''
 #[[Selecting for Tetracycline Sensitive E. coli]]
 ==Bio113 Lab Protocols==

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 # [http://gcat.davidson.edu/SynBio18/REmbedder/rembedder.html REmbedder (insert restriction site without changing amino acids)]
 # [[Common Restriction Sites]]
 '''I. Making Selective Media'''

@@ Line 69: / Line 69: @@
 # When inducing with 3OC6 (HSL), use a '''2000 fold dilution of a 10 mg/mL stock solution'''. We have dissolved in EtOH which is not the best - degrades with time. Keep this cold.
 #When growing '''thyA- cells''', add 50 µg/mL thymine to your media. Our thyA- cells have a Kan<sup>R</sup> resistant plasmid that caused the mutation. So it is best to always use kanamycin to maintain the thyA- genotype. Thymine stock solutions (4mg/mL) can be autoclaved.
-# When using theophylline with riboswitch C, make a 40-100 mM stock solution of '''theophylline''' in 50 mM NaOH. The final concentration of theophylline should be 2 mM.
+# When using theophylline with riboswitch C, make a 40-100 mM stock solution of '''theophylline''' in 50 mM NaOH. The final concentration of theophylline should be 2 mM. It works best to make LB media with theophylline in it prior to autoclaving. This way you can avoid the NaOH.
 # [http://partsregistry.org/AHL List of auto-inducers and their catalog numbers]
 # [[Synergy Machine Protocol]]

@@ Line 69: / Line 69: @@
 # When inducing with 3OC6 (HSL), use a '''2000 fold dilution of a 10 mg/mL stock solution'''. We have dissolved in EtOH which is not the best - degrades with time. Keep this cold.
 #When growing '''thyA- cells''', add 50 µg/mL thymine to your media. Our thyA- cells have a Kan<sup>R</sup> resistant plasmid that caused the mutation. So it is best to always use kanamycin to maintain the thyA- genotype. Thymine stock solutions (4mg/mL) can be autoclaved.
-# When using theophylline with riboswitch C, make a 40-100 mM stock solution of '''theophylline''' in 50 mM NaOH. The final concentration of theophylline should be 2 µM.
+# When using theophylline with riboswitch C, make a 40-100 mM stock solution of '''theophylline''' in 50 mM NaOH. The final concentration of theophylline should be 2 mM.
 # [http://partsregistry.org/AHL List of auto-inducers and their catalog numbers]
 # [[Synergy Machine Protocol]]

@@ Line 100: / Line 100: @@
 # [http://gcat.davidson.edu/SynBio13/primer/ GGA Primer Pairs Designed for You]
 # [http://gcat.davidson.edu/SynBio18/REmbedder/rembedder.html REmbedder (insert restriction site without changing amino acids)]
 '''I. Making Selective Media'''