deletions | additions       diff --git a/Methods_Bill_Siena_this_is__.md b/Methods_Bill_Siena_this_is__.md  index fb49b98..5288f22 100644  --- a/Methods_Bill_Siena_this_is__.md  +++ b/Methods_Bill_Siena_this_is__.md 
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The previously incubated falcon tube was centrifuged and the resulting supernatant was dumped, leaving only the growth pellet of the transformed cell. Induction media, containing terrific broth, kanamycin solution, and isopropyl β-D-1-thiogalactopyranoside (IPTG), was created, and resuspended the growth pellet. IPTG induces the expression of the target protein, by mimicking the inducer allolactose in the lac operon pathway. The falcon tube, containing the resuspended growth pellet and the induction media, were covered and incubated with shaking for 37 degrees C for 24 hours.  The expression cultures were lysed and centrifuged in order to clarify the lysate. The proteins were then purified using immobilized metal ion chromatography. Absorbance of the proteins at 280 nm was quantified and further assessed using SDS-PAGE.  [Purification]  ## Assay The activity of the proteins was analyzed using a UV/vis spectrophotometric assay of p-nitrophenyl-β-D-glucosideas hydrolysis. Using three columns per mutant, 50 ul of 10X diluted protein was thermo cycled for 30 min with an 8-step gradient from 30 C to 50 C. Next, 25 ul of the thermo cycled protein was transferred onto an assay plate containing 75 ul of 10X diluted pNPG. Absorbance was monitored at 420 nm every minute for 45-60 minutes  and data analysis the rate of 4-nitrophenol production in M/min was calculated using a standard curve.  [Assay procedure, short. Enough that an expert in the field could reproduce with some assumptions]