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The conditions for antibody incubations depended on the antibody being used. The optimised conditions for polyclonal antibodies are given in Table 3.1.2. The conditions for monoclonal antibodies are given below (Table 2.3.3).

Antibody Epitope name and

sequence

Block Primary

12CA5 HA (YPYDVPDYA) 1%, lOmin 1 in 750, 1% milk

9B10 Myc (EQKLISEEDL) 3%, 20min 1 in 250, 1% milk

a-FLAG (Sigma) FLAG (DYKDDDDK) 3%, lOmin 1 in 2 0 0 0 0, 1% milk

a-T7 (Novagen) T7 (MASMTGGQQMG) 3%, lOmin 1 in 10000, 3% milk

Table 2.3.3. Optimised conditions for western blotting using monoclonal antibodies.

The blocking step was performed using 20ml block at room temperature with agitation. Primary antibody incubation was in 10ml to 20ml, for 2 hours to overnight, at room temperature with agitation. Overnight incubations were used either to maximise the signal or for reason of convenience. After removal of the primary antibody with at least 5 washes in 100ml PBST, the filter was incubated with secondary antibody. 20ml of 1 in 10000 dilution in PBST with no milk was used for 45 minutes at room temperature with agitation. After 5 washes as before the filter was developed using an ECL kit (Amersham), according to the manufacturers protocol, except only 25% of the xcommended volumes of each reagent were found to be necessary. The developed filter was exposed to Kodak X-Omat film for 1 to 20 minutes, the films were processed in a Fuji developer.

2.4 - ANTIBODY PRODUCTION

ELECTRO-COMPETENT CELL PREPARATION

A llitre culture of the strain was grown at 37°C until an absorbance of 0.8 at 600nm was reached. The culture was chilled on ice for 15 minutes, then harvested by centrifugation in a cold rotor. The pellet was resuspended in llitre cold dH2 0, re-centrifuged and

resuspended in 500ml cold dH2 0, re-centrifuged and resuspended in 20ml cold water

and finally re-centrifuged and resuspended in a final volume (including cell pellet) of 2ml containing 10% final glycerol. These cells were aliquoted and frozen in liquid nitrogen.

TRANSFORMATION BY ELECTROPORATION

For transformation, 0.2cm electroporation cuvettes (Flowgen) were chilled on ice. To a cold Eppendorf tube was added up to 0.5ng transforming DNA and lOfil competent cells, which were mixed and incubated on ice for 1 minute. The cells were transferred to the cuvette, avoiding bubbles, and a pulse charge was applied using a BioRad electroporator (settings: 25/xF, 400Q, 2.5kV, resulting in a pulse of 12.5kV/cm with a time constant of 8 to 10ms). Immediately the cells were resuspended in 400/d LB

without ampicillin and the cells plated onto the appropriate selective media (up to

2 0 0/xl/plate).

ANTIBODY PRODUCTION

Briefly, fragments from the genes RAD 17, RAD24, RAD53 and M EΠwere amplified by PCR and cloned into the pET21-b vector (Novagen) (See section 3.1.). The vectors were transformed into E. coli strain FB810, a gift from Fiona Benson, using the electroporation method. An entire colony from a fresh transformant was transferred to a large scale culture, typically 400ml. Expression of the recombinant fragments was induced by the addition of IPTG. The cells were lysed under denaturing conditions, the proteins purified using an NTA-nickel agarose (Qiagen) column according to the manufacturers protocol and used to immunise rabbits (ICRF antibody production unit - Clare Hall). The resulting polyclonal sera were tested for specificity in western blot analysis using yeast extracts from WT or the appropriate delete strain, and western blotting conditions were optimised.

2.5 - YEAST METHODS

NORMAL CULTURE CONDITIONS

Yeast cultures were grown in conical flasks in YPD liquid medium at 30°C, with agitation at approximately 200rpm. The volume of culture did not exceed one quarter of the nominal flask volume to ensure adequate aeration. Solid media was YPD plus 2%

agar. Plates were allowed to set for 24 hours at room temperature before use. Diluted liquid yeast cultures were plated for single colonies using 5 to 10 sterile 4mm glass balls (Fisher) per plate. Yeast colonies were streaked out to give single colonies using sterile, sealed SOfil glass capillaries. Plates were incubated at 30°C in the dark, colonies were approximately 1mm diameter after 40 hours.

SELECTIVE MEDIA

Solid selective media was YNB with 2% agar and the required amino acids (W303 requires histidine, leucine, tryptophan and uracil as well as adenine). The carbon source was normally 2% glucose. If galactose based media was required 2% sucrose was used in addition to 2% galactose was added, to increase cell growth rate.

ARREST OF CULTURES AT DIFFERENT CELL CYCLE STAGES

To arrest cells in G1 phase a-factor was added to the growth medium at 10/xg/ml, the culture was left for at least 2 hours. Arrest was confirmed by microscopy, a fully arrested culture contained at least 95% unbudded cells. For S-phase arrest cells were first arrested in G1 as above, then the a-factor containing medium was washed off and replaced with medium containing 0.2M hydroxyurea. This was maintained for a further hour, resulting in a uniform arrest in early S phase. Arrest was confirmed by microscopy, a fully arrested culture contained at least 90% budded cells. For arrest in G2, nocodazole was added to the media at 5/xg/ml, the culture was left for at least 2 hours. Arrest was confirmed by microscopy, a fully arrested culture contained at least 85% large budded cells. If the nocodazole arrest was initially unsuccessful this was likely to be because the population contained a large proportion of petite mutants, as mitochondrial function appears to be required for efficient nocodazole arrest, for reasons that are unclear. To overcome this problem, strains that were to be arrested in this manner were first propagated on media with glycerol as the sole carbon source, in order to remove cells lacking mitochondrial function from the population.

CELL CYCLE BLOCK AND RELEASE EXPERIMENTS

A mid-logarithmic yeast culture was arrested in G1 phase with the addition of 5ptg/ml a-factor. When >90% cells were unbudded the cells were quickly washed three times in saline, then resuspended in pre-warmed medium without mating pheromone. Samples were taken immediately before the first centrifuge step (-0 minutes), immediately after the cells were returned to medium (+ 0 minutes), and at five minute intervals thereafter.

For analysis of cell morphology samples were sonicated briefly then fixed with formaldehyde. For western blot analysis samples were snap frozen on dry ice before processing. For analysis of the G1 checkpoint cells were irradiated in saline immediately before releasing from the alpha factor block.

GAMMA IRRADIATION

An asynchronous mid-log yeast culture was harvested and washed into a 50ml falcon tube. The cells were pelleted and irradiated in a ^^^Cs irradiator (CIS bio international) as a dose rate of 2.75Gy/minute. After irradiation the cells were resuspended in medium and allowed to recover for 30 minutes before harvesting for analysis. For analysis of strain sensitivity to gamma irradiation on solid media yeast cultures were applied to YPD plates, and the plates irradiated as above.

MMS TREATMENT

MMS treatment was performed by the addition of MMS to mid logarithmic cultures at a final concentration of 0.05-0.2%. Cells were incubated for up to 1 hour with the chemical, then harvested for analysis. All media solutions containing MMS, and contaminated glasswear was treated with concentrated NaOH solution to inactivate the MMS before disposal. For analysis of strain sensitivity to MMS on solid media, YPD plates were prepared containing 0.01% MMS.

UV IRRADIATION

Asynchronous cultures, or those which had been arrested, were harvested, washed into saline and irradiated using a prewarmed Philips 254nm UV-C lamp (Ultra Violet Products). The distance from the lamp was adjusted to give a fluence rate of 3J/mVs, measured on a UVP dosimeter. The cells were irradiated in large surface area containers, at a cell density of no more than 1x10^ cells/ml. 1 0cm square petri dishes were used to irradiate up to 20ml samples, 30cm square cell culture dishes for up to 100ml, and larger volumes in stainless steel baking trays. After irradiation, the cells

this wash added to the previous cells. The cells were pelleted by centrifugation and resuspended in YPD. Recovery was for 30 minutes at 30°C, before the cells were harvested for analysis. For analysis of strain sensitivity to UV on solid media cultures were applied to the appropriate plates, which were allowed to dry before irradiating as for saline suspension.