Download Determining the proportional distribution of propagons between

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.