Difference between revisions of "NanoBio: Test Induction & Purification"

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(New page: ===Overview=== After you have verified your construct, you need to make sure your plasmid yields the right protein. You are going to do a small scale crude protein extraction and compare t...)
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Revision as of 11:54, 22 July 2008

Overview

After you have verified your construct, you need to make sure your plasmid yields the right protein. You are going to do a small scale crude protein extraction and compare the uninduced cells VS induced soluble part of cells VS induced insoluble part of cells. Ideally, a lot of the right protein is in the induced soluble part.

Materials

  • Culture* (see procedure for specifics)
  • LB w/ appropriate antibiotic
  • IPTG 0.1M or 1M (or your appropriate inducer)
  • B-PER protein extraction solution

Growing up culture

  • From overnight culture
    1. Back dilute. Take 40uL saturated culture and add to 2mL LB/antibiotic. Incubate in shaker for 2-3 hours.
    2. Add IPTG. Add enough so your final concentration is 1mM. Incubate in shaker for 3-4 hours.
  • From a plate
    1. First thing at work, pick a colony and put into LB/antibiotic. Incubate in shaker for 6-8 hours.
    2. Add IPTG. Add enough so your final concentration is 0.25mM. Incubate overnight.
  • Pellet your cells. Suggested 5 min at 16G.
  • You may save your pellet at 4 C for use tomorrow or -20C if you're going to wait longer. Freezing will cause crystallization but it doesn't matter because this is just a crude analytical purification.

Extraction

asdf Arthur K Yu 19:54, 22 July 2008 (UTC)