To begin, incubate the triglycine solution at 60 degrees Celsius. Remove three RPA pellet strip tubes from the freezer. Tap the strip tubes gently against the lab bench to dislodge the white pellets.
Transfer all three pellets into a 1.5 milliliter microcentrifuge tube A.Pipette 25 microliters of nuclease-free water to rinse any remaining RPA powder in the strip tubes. Transfer the rinse solution to a new 1.5 milliliter microcentrifuge tube B.Then, add 10 microliters of the 0.57 molar triglycine solution and 10 microliters of primer mix to the master mix tube B.After vortexing and spinning down the tube, place it on ice. Next, add 28.4 microliters of the five times RPA buffer to the master mix tube B.Mix thoroughly by strong tapping or shaking multiple times.
Transfer pellets collected in tube A into the master mix tube B and invert the tube or tap the bottom of the tube with your finger several times to mix. Observe whether the solution appears homogeneous and monophasic. Place the tube on ice.
Then add 0.71 microliters of reverse transcriptase and 2.84 microliters RNase H to the master mix tube. Gently tap or invert the master mix tube up and down many times. Briefly spin down the tube before placing it on ice.
Now, aliquot 8.4 microliters of the master mix solution to 10 pre-cooled 1.5 milliliter tubes and place the tubes on ice. After aliquoting, place the tubes in a metal rack submerged in liquid nitrogen to prepare them for lyophilization. At the freeze dryer, check collector and shelf temperatures.
Now, cover open tubes in the metal rack with a sheet of cleaning white paper. Place the metal rack along with the open tubes in the freeze dryer shelf and close the shelf door. Then, select the desired program and press Start.
After 30 minutes of the secondary drying step, press stop. Release the pressure by opening the vacuum release valve. After removing the rack from the shelf, cap the tubes immediately.
