User:Susan Schultz/Notebook/Experimental Biological Chemistry/2011/09/27

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Objective

  1. Digest and transform mutated GFP
  2. Repeat biomineralization without interruptions for UV-Vis measurements

Description

Biomineralization

A 10mL solution of 0.015mM BSA and 0.25mM hydroauric acid was prepared in a 50mM Acetate buffer, pH 3.6. 1mL of this solution was place in a cuvette and set in the UV-Vis heated to 70°C. The reaction was run in the UV-Vis, and a spectra was taken every 30 minutes.

The remaining solution was placed in an oven at 80°C.


DNA Digestion and Transformation

Last week group 2 ran a PCR which mutated green fluorescent protein to contain a cysteine after the enterokinase cleavage site.

Group 2 09/20/11 The product of this PCR reaction

was confirmed to be the desired mutated protein [http://openwetware.org/wiki/User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/21

Group 2 9/21/11]. This product was digested using DnpI <http://www.neb.com/nebecomm/products/productr0176.asp>. 1μL DnpI was added to

~50μL PCR product and the mixture was left at 37°C for 1 hour. This digested the wild type unmethylated DNA. The product was then

placed on ice.

5μL of the digested PCR product was added to 40μL NovaBlue competent cells. These cells have a high transforming efficiency.

The mixture was left on ice for 30 minutes, was heat shocked at 42°C for 30 seconds and then returned to the ice for 5 minutes.

250μL of SOC media was added and the mixture was shaken at 250rmp at 37°C for 1 hour.

A LB agar plate was prepared with 100μg/mL ampicillin, and then 100μL of the mixture was spread on the plate.

The plate was incubated at 37°C overnight.

Data

Six.PNG

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Notes

This area is for any observations or conclusions that you would like to note.


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