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-004-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 The PCR Conditions were based on Becker et. al 2021. with modified denaturing and and infinite hold temperatures.

Final Profile was as follows Iniitial Denaturation: 98 C for 2 minutes Denaturation: 98C for 10 seconds Annealing: 55C for 15s Extension: 72C for 5 minutes Cycles: 30 Final Extebsion 72C for 10 minutes Infinite hold: 4C

A second profile was tested with the following conditions in an attempt to shorten the extension times, but this profile was unsuccessful based on a lack of bands present in the gel. Final Profile was as follows Iniitial Denaturation: 98 C for 2 minutes Denaturation: 98C for 10 seconds Annealing: 55C for 15s Extension: 72C for 2 minutes Cycles: 27 Final Extebsion 72C for 5 minutes Infinite hold: 4C

A more detailed optimization to try and shorten the cycle time will be run after thanksgiving