Note: for wells 2-5(DNA)/6(RNA), transfer volume needed for 96 samples into reagent matrices, pipette volumes into the wells using a multichannel pipette, and cover all wells with a plate cover foil immediately after preparation
- Clean a hood with bleach + EtOH + Eliminase, number wells from 1-5(6), UV all tools, wells, racks, tubes, and reagent matrices
- rDNAse mix in a 50 mL falcon tube according to manual (37.5 µl rDNAse + 262.5 µl rDNA buffer per sample --> for 96 samples: 28 mL rDNAse buffer plus rDNAse reconstituted in 4 mL buffer = 32 mL) (only for RNA extraction)
- 1. well (square): 200 µl lysate per sample and 200 µl RNAse-free water for negative controls (prepare this well last; when preparing, start at the top row of the well and cover empty rows with a plate foil cover, then work your way down; that way, you avoid going over open wells with tips that contain lysate (potential cross-contamination))
a.If samples are already in a 96-well plate: transfer the lysate from the plate to the well using a multichannel pipette; make sure to follow a premade template with 1 negative control per row (adding up to 12 negative controls)
b.If samples are in tubes: transfer samples to PCR 12-strip tubes following a premade template, add negative controls accordingly (one per row), just prep one row at a time and transfer the lysate using a multichannel pipette
- 2. well (square): 650 µl buffer MWA3 per sample (note: protocol says 850 µl but well would overflow) (x 96 = 62.4 mL; when prepping fill reagent matrix to 25 mL, then refill to 25 mL, then refill half)
- 3. well (square): 650 µl buffer MWA3 per sample (x 96 = 62.4 mL; when prepping, fill the reagent matrix to 25 mL, then refill to 25 mL, then refill half)
- 4. well (square): 650 µl buffer MWA4 per sample (x 96 = 62.4 mL; when prepping, fill the reagent matrix to 25 mL, then refill to 25 mL, then refill half)
- 5. well (300µl well): 100 µl water per sample (x 96 = 9.6 mL; when prepping, fill 10 mL into reagent matrix)
- 6. well (square): 300 µl rDNAse mix per sample (x 96 = 28.8 mL; when prepping, fill the reagent matrix to 25 mL, then refill the rest of the rDNA mix into the same reagent matrix)