NanoBio: Test Induction & Purification

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)