Team:UCLA/Notebook/Honeybee Silk/11 August 2015

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Ligation of psb1c3+T7 promoter and Silk+GFP

Component	Volume (out of 20uL)
T4 Ligase Reaction Buffer	2 ul
Backbone (psb1c3+t7 promoter)	0.8uL (50 ng)
Insert (silk + spyC)	1.5 ul (146 ng)
H20	14.7 ul
Ligase	1uL

• Program =16C overnight, 10 minutes heat inactivation at 65C.

8/11

Protein Yield

• Yield from 8/7 silk purification
• BCA assay
  • Based on the interpolated Absorbencies from the BCA assay the concentration of our 8/7 silk is approximately 0.78 mg/ml in around 12 ml of solution, giving a conservative 9 total mg of protein for a 300 ml culture.
• This is an improvement over the 7/29 purification where we got around 1.6 mg/ml in only 0.75 ml of solution from a 100 ml culture.