In vitro Testing of pT7-GFPmut3b Construct
To determine if the pT7-GFPmut3b(INSERT LINK) construct works in vitro. In addition we wish to characterize this constructs response at varying temperatures.
Materials and Methods
Refer to protocols page.
Tested on (Insert link)
- Negative Control-pT7 construct (LINK)
- DNA Concentration - 4µg
- Total Volume - 60µl
Figure 1. shows us that the pT7-GFPmut3b synthesizes over time for all the temperatures tested. When the three temperatures are compared it is clear that 4°C is the minimum temperature for expression and that 25°C is the maxiumum. Interestingly 37°C is not the maximum for pT7 in vitro, with 25°C producing almost double the molecules of GFPmut3b at 37°C.
All three temperatures appear to stop synthesis at a similar level. Although it is difficult with such limited sampling to extract the exact time the synthesis stops, we can approximate it to greater than 200 minutes.
The variation within the samples was different for each temperature. 4°C and 25°C show limited variation with 37°C showing the greatest variation.
To conclude the following approximations can be made:
- Synthesis stops >200 minutes
- 4°C - 1.2x1013 molecules of GFPmut3b produced
- 25°C - 4x1013 molecules of GFPmut3b produced
- 37°C - 8.3x1013 molecules of GFPmut3b produced
- 25°C was the optimum temperature that we found