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 stability 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: GM3F.Universal : AGA GTT TGA TCM TGG C

Reverse Primer: GM4R.UNIVERSAL : TAC CTT GTT ACG ACT T

Using the samples t4-t7 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 A varying matrix of PCR conditions was tested with the following variations in Profile

Iniitial Denaturation: 98 C for 2 minutes Denaturation: 98C for 10 seconds Annealing: 51.3C, 53.1 C and 55C at 30, 60 and 90 and 120s Extension: 72C for 30, 60, and 90s Cycles: 30 Final Extebsion 72C for 2 minutes Infinite hold: 4C Annealing temperatures were tested using a gradient at each of the different annealing times first with an extension time of 30s the optimal conditionfor annealing were determined to be 55C for 30 seconds, at which point two more PCRs were ru to determine the optimal annealing time

Final Profile was as follows Iniitial Denaturation: 98 C for 2 minutes Denaturation: 98C for 10 seconds Annealing: 55C for 30s Extension: 72C for 90s Cycles: 30 Final Extebsion 72C for 2 minutes Infinite hold: 4C

All Reactions were checked using a 100ml 1% agarose gel containing 5ul of Sybr safe, which was run at 110V for 85 minutes