In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we describe an optimized conjugation protocol for the horizontal transfer of ICE or chromosomal DNA carrying antibiotic resistance markers (i.e., tetracycline, gentamicin, puromycin) from donor to recipient mycoplasma cells. Calculation of the conjugation frequencies, selection and characterization of transconjugants are detailed. This protocol has been developed with M. agalactiae but has been successfully used for M. bovis and can be adapted to

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this