TITLE: General bacteria and archaea 16S-rRNA (515Fmod-806Rmod) for Illumina amplicon sequencing
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Universal 16S rRNA probe-based-qPCR assay for bacteria.</div><div class = "text-block"><span>The primers target the V4 region of the 16S rRNA gene and were specifically designed for Illumina amplicon sequencing. The original primers were designed by Caporaso </span><span style = "font-style:italic;">et al.</span><span> (2012) and modified by Walters </span><span style = "font-style:italic;">et al.</span><span> (2015). For barcoding, we use the </span><a href="https://www.fluidigm.com/products/access-array" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Fludigm Access Array</span></a><span> for barcoding the sample and therefore the primers are synthisized with the CS1 and CS2 regions.</span></div></div>

[STEPS]
?. [Primers]
ABCD1NameDirectionSequence1Target region22515Fmod_CS1ForwardACA CTG ACG ACA TGG TTC TAC AGT GYC AGC MGC CGC CGT AA515-5333806Rmod_CS2ReverseTAC GGT AGC AGA GAC TTG GTC TGG ACT ACN VGG GTW TCT AAT787-8061. CS + primer sequence (in bold)2. Relative to E. coli SSU rRNA gene
ABCD1NameDirectionSequence1Target region22515Fmod_CS1ForwardACA CTG ACG ACA TGG TTC TAC AGT GYC AGC MGC CGC CGT AA515-5333806Rmod_CS2ReverseTAC GGT AGC AGA GAC TTG GTC TGG ACT ACN VGG GTW TCT AAT787-806
?. [PCR reaction]
Prepare the following master mixture .Do not forget to prepare some additional mixture for the negative (NTC) and positive controls, and to account for pipetting errors.   ABCD1ReagentFinal. conc.1 tube (25 μl)100 reactions (96-well plate; µl)2PCR H2O
&nbsp;17.5251752.5310X DreamTaq Green Buffer1X2.52504dNTP (2 mM each)0.2 mM2.52505BSA (20 μg μl-1)80 ng µl-10.1106515Fmod-CS10.2 μM0.62562.57806Rmod-CS20.2 μM0.62562.58DreamTaq Green DNA Polymerase0.625 U0.12512.59Final volume&nbsp;242400
on ice
ABCD1ReagentFinal. conc.1 tube (25 μl)100 reactions (96-well plate; µl)2PCR H2O
&nbsp;17.5251752.5310X DreamTaq Green Buffer1X2.52504dNTP (2 mM each)0.2 mM2.52505BSA (20 μg μl-1)80 ng µl-10.1106515Fmod-CS10.2 μM0.62562.57806Rmod-CS20.2 μM0.62562.58DreamTaq Green DNA Polymerase0.625 U0.12512.59Final volume&nbsp;242400
?. [PCR reaction]
Vortex and spin down
?. [PCR reaction]
Distribute   of the mixture to each tube and add   of template DNA or cDNA
24 µl
1 µl
?. [ PCR reaction]
Run the following PCR program:    2.1        2.2        2.3    3. 4.  hold
94 °C
94 °C
52 °C
72 °C
72 °C
4 °C
?. [Evaluate PCR products on an agarose gel]
Prepare a 1.5% agarose gel by mixing:Heat in the microwave until dissolved and pour into a gel frame.Place solid gel into an electrophoresis bath filled with TAE buffer.
[TAE]
[agarose]
?. [Evaluate PCR products on an agarose gel]
Mix up to  of the PCR reaction sample with  of loading dye and load the sample into a well.In addition load  of DNA ladder mix (80-10,000 bp) into an empty well, as a marker.
5 µl
1 µl
5 µl
?. [Evaluate PCR products on an agarose gel]
Run the gel at 110V, 265mA for approx.
?. [Evaluate PCR products on an agarose gel]
Stain gel for at least 40min in an Ethidium bromide TAE bath (or any other DNA stain).
?. [Evaluate PCR products on an agarose gel]
Visualise the gel using a gel documentation system.