IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.2

STATUS: PROTOCOL UNDER CONSTRUCTION

Experiment 1

Aims:

• To create a series of dilutions of GFP using a purified sample of GFP

Status: FEEDBACK/VALIDATION REQUIRED

• Purified GFP is to be ordered from clone tech at the moment waiting for a quote from clone tech

Reagents

• Recombinant GFP (Mw=27,000Da) Link to product information. It is in the following form;
• 100ug in buffer of 1mg/ml, this means in the solution there is:
  • 3.7x10^-9 moles of GFP
  • 0.037 M (moles dm^-3)
  • Volume supplied to us is 0.1mL

Protocols

• The moles of GFP that our in vitro systems is expected to produce is 7.4x10^-12 moles, this is based upon the quoted expected protein production of 200ng (2x10^-7/27000Da). If the total volume of the cell extract is 100ul, this means that the concentration is around 7.4x10^-8 M.
• We need a sufficient range of dilutions to cover this range. However, the relationship between fluorescence and molcules of GFP is linear at concentrations in uM and lower. Therefore, if we have a sufficient range we should be able to plot an accurate calibration curve.
• We propose covering the following range of 8 data sets with 2 repeats for each:
  

1.000uM
0.500uM
0.250uM
0.125uM
0.060uM