Objectives:

To optimize PCR conditions for the Sequencing primers for 16s rRNA amplification using the Q5 mastermix Kit from NEB

Broader Context

These primers will initially be used to sequence and analyze members and stabilit of the microbiome as part of PRJ-003-Aiptasia-Stab, but will also be utilized for other sequencing projects in the lab

Protocol

Forward Primer: 515FB.A501 : AAT GAT ACG GCG ACC ACC GAG ATC TAC ACA TCG TAC GTA TGG TAA TTG TGT GYC

Reverse Primer: 806RB_SA701 : CAA GCA GAA GAC GGC ATA CGA GAT AAC TCT CGA GTC AGT CAG CCG GAC TAC NVG

Using the samples t4-t6 a extracted during lysis matrix optimization, and a water blank we performed 25 ul reactions with the following ratios Q5 mastermix (2x) - 12.5ul 10uM forward Primer - 1.25ul 10 uM reverse primer - 1.25ul Template DNA - 1ul Nuclease free water - 10 ul

Profile was taken from previous run

• Iniitial Denaturation: 98 C for 2 minutes
• Denaturation: 98C for 10 seconds
• Annealing: 55C for 15s
• Extension: 72C for 5 minutes
• Cycles: 30
• Final Extension 72C for 10 minutes
• Infinite hold: 4C however Extension times were also run at 3 and 4 minutes.

the results eem promising however it also appears there was some contamination in the reagents causing an extra band to form, i will rerun with different mastermix and update.