IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1: Difference between revisions
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=='' | ==''Infector Detector''== | ||
===1)Initial testing=== | ===1)Initial testing=== | ||
'''Construct:''' pTet - LuxR - pLux - GFP <br> | '''Construct:''' pTet - LuxR - pLux - GFP <br> |
Revision as of 02:04, 14 August 2007
Infector Detector
1)Initial testing
Construct: pTet - LuxR - pLux - GFP
Test to see if construct will express in vitro. Experiments carried out under room temperature 25oC and under inducer concentration that was shown to give a high induction in vivo.
Aims:
- To determine if construct expresses in vitro
- To get approximations of: life span, response time and rate of GFP
- To determine whether the constructs or the in vitro need to be optimised.
Constant Conditions:
- 25 oC
- 50μl in vitro chassis
- DNA added 2μg
- AHL concentration 1000nM
Variables:
- Independent variables :
- Dependent variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
Sampling:
- Every 5 minutes.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system with no AHL added
- Positive Control: In vitro system with purified GFP added
Protocol
Equipment
- Incubator 30oC
- Fluorometer
- Plate x1
- Plate Centrifuge
- 6x1.5ml tubes
- Gilson Pipettes
- Stop watch
Preparations of Chemicals and Reagents
After preparing all solutions preheat to 30oC
- Remove .... of our cell extract solution from storage solution
- Now we need to prepare 50μl of the commercial E.coli and T7 cell extract solution. First we need to prepare complete amino acid mixture for both extract solutions: Add the 7.5μl volume of two amino acid minus mixtures for both kits
- Take 3x1.5ml tube and add 5µl of the E.coli complete amino acid mixture
- Take 3x1.5ml tube and add 5µl of the T7 complete amino acid mixture
- To each 1.5ml tube add 20µl of S30 Premix Without Amino Acid
- Add 15µl of S30 Extract Circular
- Add ...nuclease-Free Water
Loading Plate
- Follow the schematic for the plate adding everything but the DNA sample.
- Place the top on the plate and place in the incubator. Leave for a few minutes to heat to 30oC
- Remove from incubator and centrifuge for 1 minute
- Remove lid and Measure in the flourometer.
- Then to begin the reaction add .... purified DNA sample.
- Place lid back on and place back in the incubator at 30oC
Collecting Data
- After 5 minutes of incubation measure the fluorescence by repeating procedure 3-4 above.
- Repeat measurements after every 5 minutes until the Fluorescence is constant
- Before every measurement in the fluorometer spin the plates in a plate centrifuge
Schematic
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