ARISA

Primers

Use "primers ITSF (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITSReub (5′-GCCAAGGCATCCACC-3′)", which are, " respectively, complementary to positions 1423 and 1443 of the 16S rRNA and 38 and 23 of the 23S rRNA of E. coli." [1]

Sources:

ARISA Protocol

Version 1

Note: this protocol is Copied from https://academic.oup.com/femsec/article/85/3/483/583834#95229670 Diversity of the skin microbiota of fishes: evidence for host species specificity.

1. Extracted DNA was used as a template for PCR on the internal transcribed spacer region using the ITS-FEub (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITS-REub (5′-GCCAAGGCATCCACC-3′) primers (Cardinale et al., 2004).
2. Ribosomal internal spacer analysis (RISA) was performed as previously described by Arias et al. (2006) with the following modifications. The PCR mix contained 1× Taq buffer, 0.4 mM dNTPs (Promega, Madison, WI), 0.4 μM ITS-FEub primer, 0.2 μM ITS-R primer, 0.02 μM ITS-REub labeled primer, 5 mM MgCl2, 1 U of Taq polymerase (5 PRIME, Inc., Gaithersburg, MD), and 100 ng of template DNA in a final volume of 50 μL.
3. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 55 °C for 1 min, and 68 °C for 2 min, ending with a final extension at 68 °C for 7 min.
4. Ten microliters of each PCR product was diluted with 5 μL AFLP® Blue Stop Solution (LI-COR). Diluted samples were denatured at 95 °C for 5 min followed by rapid cooling prior to gel loading to prevent reannealing.
5. PCR products were electrophoresed on the NEN Global Edition IR2 DNA Analyzer (LI-COR) following manufacturer's instructions. One microliter of sample was loaded into each well.

Sources: https://academic.oup.com/femsec/article/85/3/483/583834#95229670 Diversity of the skin microbiota of fishes: evidence for host species specificity https://aem.asm.org/content/70/10/6147 Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities

Note to future researchers: As the first phase of this experiment was not able to test ARISA for use in the experimental system, experimentation regarding the efficacy of ARISA to measure community composition is needed.