Began my full QuantSeq library prep today. I am processing ctenidia samples first, and since there are 53 samples I’m doing ~half at a time. Today I generated double stranded cDNA for 26 samples + 2 NTC (28 total). I loaded samples onto a PCR plate in 4 rows of 7.

Samples processed, volumes used for RNA and DEPC-treated water, and total RNA used.

Volumes of solutions needed - I aliquoted volumes into 7 pcr tubes, so I could then add to samples using a multichannel pipette.

PCR Plate setup

Notes

• Samples were thawed on wet ice, and vortexed once before use.
• I loaded the first 3 samples, 328, 299 and 301, onto the PCR plate but then had to wait a few minutes (~3-5) for the rest to thaw.
• Step #2: held samples at 42C for 14 minutes while preparing master mix. Probably a bit too long.
• I used a whole PCR Plate, which took up the entire thermocycler space. Next time, I should use partial PCR plates to leave room for a PCR strip to pre-warm the master mix needed for step 3 & 4.
• The centrifuge gradually cooled as I was using it, which was weird since I didn’t change the temp. I think I need to set temperature to 22C every time I use it, b/c it may default to 4C.
• I aliquoted 20uL of each sample to new tubes and placed in -80 freezer.