Replication timing by comparative hybridization

R-KT-DIR ) or 4 hr ( R-Zeo-DIR ), then add dry glucose (2% w/v final concentration). Thirty minutes later, shift the culture to the 37^o bath.

5. Once the culture has reached 37^o C (it takes 10-15 min), add Pronase (0.01-0.05 mg/ml final conc) to release the cells from the alpha factor block. [You can add dry Pronase. I usually dissolve the Pronase in 5 ml of minimal medium, just to avoid having the dry Pronase stick to the sides of the flask.]

6. Hold at 37^o C for 90-120 min (until budded cells accumulate). Remove one sample (t = 0), then swirl the flask in ice-water and return it to 23^o C. Continue to collect samples every 4 min (until t = 80 min or more). I usually take two 0-min samples, and take samples every 4 min from t=8 through t=56, then take t=64, 72, and 80 min samples--19 samples in all.

The ice-water treatment is to quick-chill the culture to 23^o C, and is usually for ~1 min. You can empirically determine how long it takes to bring your desired volume of medium from 37^o to 23^o C.

Collecting samples : Freeze 8 ml of 0.1% NaN3, 0.2 M EDTA in "50 ml" Oakridge-type plastic, screw-capped centrifuge tubes (frozen slanted to maximize the surface area). During the experiment, mix 20 ml of cells with 1/50 volume of 10% NaN₃ in a 35 ml Corex tube (on ice) and immediately transfer the mix to a tube of frozen EDTA/NaN₃ . Alternatively, squirt the 10% azide on the frozen azide/EDTA and immediately add the cell sample. Vortex or shake the tube vigorously to chill the sample and break up the frozen EDTA. Spin down the cells in a chilled centrifuge, wash with 1 ml cold water per sample in Eppendorf tubes and freeze the pellets at -20^o C.

7. Extract the DNA ( smash-&-grab with glass beads); digest 1/4 to 1/3 of it with the enzyme of choice in 40-60 microliters (EcoRI works well, as it cuts the ARS-less circle just once). Split each digest in two or three to run in duplicate (or triplicate) gels. It helps to make wide, well-separated wells. Blot the gels as usual.

8. Hybridize simultaneously with probes for the ARS-less circle, an early marker, a late marker, and the sequence of interest. Quantitation comes out best if the fragments to be probed are in the 2 kb-8kb range. Sometimes the fragments just don't work out (i.e., two fragments are too close for comfort), in which case you'll have to strip the blots and re-probe. If so, I usually include the ARS-less circle probe for each hybridization.

9. Quantitation: we use a Molecular Dynamics PhosphorImager or a Packard InstantImager. The values obtained for the two t=0 samples are combined and used as the zero time point (to be used for normalization of the later samples).

Here's a typical plot showing replication of ARS1 and R14 (a fragment near the right end of chromosome V):