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In situ hybridisation on yeast with Locked Nucleic Acid in situ detection probesProtocol prepared by Dr. Torben Heick, Department of Molecular Biology Aarhus University, Denmark The protocol originates from the Singer laboratory (Long et al. (1997) Science 277, 383-387) and has been modified for yeast, Saccharomyces cerevisiae, cell cultures subjected to a heat-shock treatment. However, the outlined routines for fixation etc. should be applicable for yeast cell cultures in general.
Ref.: The protocol is modified from Thomsen et al. (2005) RNA, 11:1745-1748.
es (10 mL) were grown at 25ºC to an OD600 of 0.1-0.3 and subjected to an instantaneous temperature-shift • er incubation at the desired temperature and time, e.g. 31ºC for 90 min, cells were fixed for 15 min at the experimen- tal temperature by adding formaldehyde to a final concentration of 4%. This was followed by 30 min incubation at 20ºC.
Fix • ed cells were pelleted by centrifugation and washed three times in 1 ml wash buffer (1.2M sorbitol, 0.1M KH PO / K HPO4, pH 6.5), and subsequently resuspended in 200-500 μL wash buffer.
ell suspensions were plated on 14-well, 0.1% poly-L-lysine coated glass slides (Immuno-Cell Int.), washed in wash buffer containing 1% ß-merchaptoethanol before spheroplasting for 10-15 min at 30ºC in 10 μL oxaly- ticase solution (20mM vanadyl-ribonucleoside, 0.2% ß-merchaptoethanol, 0.1 U/μL RNasin, 1.2M sorbitol, 0.1M KH PO / K HPO , pH 6.5, 0.1 mg/mL oxalyticase (Enzogenetics)).
y, cells were washed for 5 min at 20ºC; twice in wash buffer, once in (0.1M KH PO /K HPO , pH 6.5, 0.1% NP40) and once in (0.1M KH PO /K HPO pH 6.5), and finally incubated with cold 70% ethanol for 15-30 min at -20ºC.
• obe preparation was done by direct labelling of 10-20μg of 5’-end amino modified Locked Nucleic Acid in situ hybri- dization probe with 300μg of monofunctional Cy3 NHS-Ester (Amersham Pharmacia) in 0.1M NaHCO /Na CO , pH 9.0 overnight at 25ºC in the dark. The probe was purified on a G-50 spin column and the concentration determined by OD measurement.
F • or each well, 100ng of purified probe was mixed with 10μg salmon sperm DNA and 10μg yeast tRNA, lyophilized and resuspended in 5μL solution I (80% formamide, 10mM NaHPO pH 7.0), denatured for 5 min at 95ºC and finally mixed with 5μL of solution II (4xSSC, 20mM Vanadyl-ribonucleoside, 4μg/μL BSA, 0.1U/μl RNasin).
e, cells were drained for ethanol and washed in 10-20 μL (depending on well size) at 20ºC, twice for 5 min in 2xSSC and once for 10 min in 40% formamide/2xSSC, 0.1% Triton X-100, before overnight incubation at 37ºC with 10 μL of probe mix.
Remo val of probe solution was followed by washing steps in 10-20 μL volume: (i) twice in 40% formamide/2xSSC for 10 min at 37ºC, (ii) once in 2xSSC/0.1% Triton X-100 for 10 min at 20ºC, (iii) twice in 1xSSC for 10 min at 20ºC, and (iv) twice in 1xPBS for 5 min at 20ºC.
Finall y, 2.5μl of mounting solution (1xPBS pH 8.0, 80% glycerol, 0.1μg/mL DAPI) was applied to air-dried wells, which were subsequently covered with a cover slip and analysed by fluorescent microscopy.
NB: Optimisation of the standard conditions by increasing hybridization stringency conditions can be achieved by increa-sing the formamide concentration (normally 40%) in hybridization- and all appropriate wash-buffers to 50%, 60% and 70%, respectively.
ImagingImages were acquired on an Olympus BX51 microscope equipped with cooled Olympus DP50 CCD camera and analysis software.
Buffers*Please note: For optimal fixation it may be critical to use fresh formaldehyde solutions. Fresh 4% solutions can be made from 16%, methanol free, formaldehyde or from solid paraformaldehyde (4% w/v).
For preparation of buffers please refer to :Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. --3rd ed. -- New York: Cold Spring Harbor Laboratory, 2001.
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