This post details information on ITS2 clean-up for the my ambient Mcap developmental timeseries samples from 2023 and for the POC spawning experimental samples from 2023. Mcap 2023 github repo is here and POC github is here. See Ariana’s post about the ITS2 protocol for this project.

Samples

Samples (44 total samples) were amplified on 5/13/24 and 5/14/24, but there appeared to be some primer dimer present, as evidenced by bands ~100bp. Therefore, we decided to do a bead clean-up on all samples.

Equipment and materials

• Kapa Pure Beads
• Tris-HCl (10 mM)
• Molecular grade ethanol
• Magnetic stand
• PCR tubes
• Gel rig + materials

Protocol

Protocol was followed according to this post. A few notes:

• After adding Kapa Beads, I incubated tubes for 15 minutes at room temperature.
• After the ethanol washes, I dried beads for ~4 minutes until beads were matte/ethanol had evaporated.
• After adding the elution buffer (10 mM Tris-HCl), I incubated tubes for 8 minutes at room temperature.

QC

Following the clean-up protocol, I ran a 2% gel for 90 minutes at 100 volts + 100 amps to check if the primer dimers were removed. I also used a 1kb ladder and a 100 bp ladder. The machine that the gel rig is hooked up to has only been reaching ~70-80 volts.

Gel looks great! No primer dimer from what I can see. Hollie will send to Janet to confirm if these are good enough for sequencing.