Testing DNA concentration of pTet-GFPmut3b In vitro
To determine if the optimum concentration of pTet-GFPmut3b in vitro.
Materials and Methods
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- Negative Control- Nuclease Free Water was added instead of DNA
- Temperature - 37°C
- Total Volume - 60µl
Figure 1.1 and Figure 1.2. show us that the synthesis of GFPmut3b increases with DNA added. Though the molecules of GFPmut3b synthesised increases with increasing DNA concentration, even after a concentration of 4µg. This is not what was expected because of the nature of the cell free chassis which is optimum DNA concentration = 4µg. But the increase in fluorescent moecules synthesised by increasing DNA concentration is not very big. If DNA concentration is increased from 2µg to 6µg (a percentage increase of 200%), it reuslts in only about 15% increase (at 360min) in GFPmut3b molecules synthesised. Thus it was decided that a DNA concentration of 2 or 4&micor;g will be used for testing pTet-GFPmut3b construct.
To conclude the following approximations can be made:
Optimum DNA concentrations - 2 or 4µg of DNA