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Determining the proportional distribution of propagons between mother and daughter cells This method is that described by Cox et al. (2003) with minor modifications. 1) Using a Singer micromanipulator (or equivalent), separate individual cells onto solid YPD media containing 5mM GdnHCl. At this concentration of GdnHCl, propagon replication is inhibited and the numbers of propagons the cells contain (n0) is fixed. 2) Incubate the cells at 30o C for approximately two hours until the cells have undertaken one round of cell division. 3) Separate the resulting daughter cell from the mother by micromanipulation to fresh solid YPD media containing 5mM GdnHCl. Incubate both mother and daughter cells at 30o C for 48h to allow them grow into small colonies. As these colonies are grown in the presence of 3mM GdnHCl, propagon replication remains inhibited within the cells of the colony, so that after 48h (twenty generations = approx. 106 cells) there have been sufficient divisions to allow the segregation of each original propagon to a separate cell. 4) Pick these small colonies and re-suspended them in sterile phosphate buffered saline before plating onto solid adenine drop-out media supplemented with 5% (v/v) YPD. Only adenine-independent [PSI+] cells will grow on this media. Count the number of resulting white colonies, which is a direct estimate of the number of propagons the original cell contained. The total number of propagons (n0) is therefore the sum of the number of colonies on the plates derived from both the mother and daughter cell, with the proportion of propagons (π) being the fraction derived from the daughter cell (Cole et al, 2004). Media and Solutions YEPD liquid media 1 % (w/v) yeast extract, 1 % (w/v) bactopeptone, 2 % (w/v) glucose). Adenine drop-out media 2 % (w/v) glucose 0.67 % (w/v) yeast nitrogen base with ammonium sulphate 0.2 % (w/v) adenine drop-out mix (Formedium, UK) 2 % (w/v) granulated agar 3M GdnHCl stock per 100ml: 286.6g guandine hydrochloride in 100ml H2O Sterilise by autoclaving. Phosphate Buffered Saline (PBS) per litre: 8g NaCl 0.2g KCl 1.44g Na2HPO4 0.24g KH2PO4 Adjust pH 7.4 and sterilise by autoclaving.