*To determine if construct expresses in vitro at temperatures of: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
*To determine how long the system can last (when GFP is no longer produced) at each temperature.
*To determine the maximum rate of GFP produced at each temperature range by taking fluoresence reading over a period of time ( time frame to be determined by Friday 17th August)
===Status===
???
===Equipments===
*Fluorometer + PC
*Fridge at 4oC
*Water bath in cold room at 15oC
*30oC water bath
*37oC shaking incubator
*50oC heating block
*6 Fluorometer plates (black)
*6 Sealing plate mats
*Gilson pipettes 1000, 200 and 20
*Eppendorf Tubes
*Plate Centrifuge
*Stopwatch
===Reagents===
__NOTOC__
*Our Prepared S30 extract: Optimised amount for experimentation to be decided after experiment 2.1
==Aims==
*Commercial S30 E.coli extract. Including:
To investigate the fluorescence dependence of varying media volumes to no. of moles of GFP. This is to determine the need for the maintenance of a constant volume throughout subsequent experiments, as with making GFP dilutions for the calibration curve.
**175µl Amino Acid Mixture Minus Cysteine, 1mM
**175µl Amino Acid Mixture Minus Methionine, 1mM
**175µl Amino Acid Mixture Minus Leucine, 1mM
**450µl S30 Extract, Circular (3 × 150µl)
**750µl S30 Premix Without Amino Acids
*Commercial S30 T7 extract. Including:
**175µl Amino Acid Mixture Minus Cysteine, 1mM
**175µl Amino Acid Mixture Minus Methionine, 1mM
**175µl Amino Acid Mixture Minus Leucine, 1mM
**450µl T7 S30 Extract, Circular (3 × 150µl)
**750µl S30 Premix Without Amino Acids
*MiiA water x1ml
*GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)
===Steps===
#First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
#Place each of the 96 wellplates in their respective incubators so as to heat them up to the appropriate temperature before the experiments start.
#''Commercial E.coli Cell Extract'': First prepare a complete amino acid mixture for both extract solutions: Add the 90μl volume of two amino acid minus mixtures into an labelled eppendorf to give a volume of 180μl. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench.
#''Commercial T7 Cell Extract'':Carry out procedure above for the T7 amino acid minus mixtures, but using 45μl of the two amino acid minus mixtures each to make up to a total volume of 90μl.
#''S30 home-made Cell Extract'':Remove XXXμl and place in a labeled eppendorf tube. Place these tubes in a rack on bench.
#''Commercial E.coli Cell Extract'':Take a eppendorf tube and add 5µl of the E.coli complete amino acid mixture. Then add 20µl of S30 Premix Without Amino Acid. Then add 15µl of S30 Extract Circular. Finally add nuclease-Free Water to bring final volume (inc.DNA vol) to 100µl, the volume of DNA added will be determined in experiment 1 and the volume of the nuclease free water adjusted accordingly. Place the eppendorf tube in a rack on the bench
#''Commercial T7 Cell Extract'':Repeat step 5 however use the T7 cell extract reagents and the T7 prepare complete amino acid mixture. We will now have two eppendorf tubes of prepared commercial T7 cell extract.
#Vortex the tubes to mix thoroughly and place the 5x eppendorf tubes in the incubator at 30<sup>o</sup>C
'''Loading Plate'''
==Materials and Methods==
#Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
Refer to protocols page.
#Place the lid on the 96well plate and put into the incubator at 30<sup>o</sup>C for 10 minutes to allow temperature to equilibrate
#Remove from 30<sup>o</sup>C incubator and spin-down in centrifuge in plate centrifuge at 2000rpm for a few seconds. Spin down is the process of bringing down any solution on lid or side of well into the base of the well. Alternatively can tap the top of the lid to bring down any solution to bottom of the well.
==Results==
#Remove lid off th e 96well plate and place in the fluorometer. Create a file name protocol 2-1 under: D:\IGEM\'''INSERT DATE'''\CBD\ protocol 2-1. Export the data here. If repeated measurements change the second number to suit repeat number, e.g. 2nd repeat protocol 2-2, 5th repeat protocol 2-5. Once the data collection is set up then initiate the measurements.
[[Image:IC2007 Experimental Design GFP Dilution in water.PNG|thumb|left|800px| Fig.1: Fluorescence of varying media volume]]
#This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
<br>
#Then to begin the reaction add required volume of purified DNA sample to give 2µg to the wells indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
#Place lid back on and place back in the incubator at 30<sup>o</sup>C <br><br>
Interestingly, the addition of more media (water) increased the fluorescence readings of the samples to a significant extent (Fig.1).
#After 5 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. This initial measurement of 30 minutes is to find out how fast GFP is being produced. After this initial measurement, the intervals should be reassessed and adjusted accordingly
#Before each measurement be careful to remember to either spin down or tap down the solution and to remove the lid before placing in the fluorometer
==Discussion==
Fig.1 suggests that fluorescence does depend on the wolume of media to which the substrate is diluted in, albeit in a proportionate way. While one would expect the opposite to occur, the results suggest that volume of media, and hence the maintenance of the same volume through subsequent experiments, is vital for an accurate reading. Indeed this also implicitly implies the importance of minimizing evaporation in our media.
==Conclusion==
* Constant volume must be maintained for all experiments.
* Evaporation (reducing media volume) may play a huge role in experimental readings.
Latest revision as of 04:04, 16 October 2007
Fluorescence Dependence on Media Volume
Aims
To investigate the fluorescence dependence of varying media volumes to no. of moles of GFP. This is to determine the need for the maintenance of a constant volume throughout subsequent experiments, as with making GFP dilutions for the calibration curve.
Materials and Methods
Refer to protocols page.
Results
Fig.1: Fluorescence of varying media volume
Interestingly, the addition of more media (water) increased the fluorescence readings of the samples to a significant extent (Fig.1).
Discussion
Fig.1 suggests that fluorescence does depend on the wolume of media to which the substrate is diluted in, albeit in a proportionate way. While one would expect the opposite to occur, the results suggest that volume of media, and hence the maintenance of the same volume through subsequent experiments, is vital for an accurate reading. Indeed this also implicitly implies the importance of minimizing evaporation in our media.
Conclusion
Constant volume must be maintained for all experiments.
Evaporation (reducing media volume) may play a huge role in experimental readings.
