In a 1.5 milliliter microcentrifuge tube on ice, combine 10 microliters of the cell-free extract with four micrograms of plasmid DNA and 25 microliters of 2X cell-free protein synthesis buffer. Make up to a total volume of 50 microliters with double-distilled water to prepare the cell-free protein synthesis solution. Weigh 0.75 grams of agarose and add it to 100 milliliters of double-distilled water buffer to prepare 0.75%agarose.
Microwave the 0.75%agarose in 30-second bursts at high power and pipette 50 microliters of the molten agarose into 1.5-milliliter microcentrifuge tubes or into molds of a desired shape. Place the molten agarose on a heat block set to 50 degrees Celsius and allow the agarose to cool but not polymerize. Then, mix the molten agarose with the cell-free protein synthesis solution by pipetting and stirring with the pipette tip.
Allow the gels to cool to room temperature and polymerize for approximately two minutes. Transfer the polymerized agarose to 1.5-milliliter microcentrifuge tubes with a spatula and flash freeze in liquid nitrogen. Place the flash-frozen hydrogels into minus 80 degrees Celsius storage for one hour.
After that, remove the microcentrifuge tube lids. Cover the tubes with a wax film and pierce the film to allow the moisture to be dried off. Set the temperature of the freeze dryer to minus 20 degrees Celsius and pressure to 0.1 millibars and freeze-dry the cell-free protein synthesis devices for 18 hours or overnight.
Rehydrate the freeze-dried devices for 30 minutes with 50 microliters of double-distilled water without excess liquid, and transfer the gels to a black 384-well microtiter plate using a spatula. Finally, place the microtiter plate on a plate reader and use the plate reader settings shown on the screen for fluorescence detection and analysis. The cell-free protein synthesis of eGFP and mCherry in a hydrogel using E.coli cell lysates is shown in this figure.
0.75%agarose gels were prepared without DNA template with four micrograms of either eGFP or mCherry template or with four micrograms of both eGFP and mCherry template. An overlay of the two channels is also shown, and the overlay includes the differential interference contrast image. The results confirmed the co-expression of both mCherry and eGFP in agarose.
