Replication timing using transient hemimethylation

Tuesday, November 18, 2003

Description
Replication timing using transient hemimethylation

Procedure
1. Grow ~330 ml of RM14-3a expressing dam methylase (RM14-3a Meth5) to an OD660 of about 0.3 (3.85 x 106 cells/ml) at 23oC. Arrest with 200 nM alpha factor for 1-1.25 doubling times, until the percent budded cells stops increasing (usually 395%). (RM14-3a is bar1 , so the alpha factor concentration required is low).

2. Transfer cells to 37oC and wait for culture itself to reach 37oC. Add Pronase (20 microgram/ml final concentration -- dissolve Pronase in 5 ml minimal medium). We use Pronase from Calbiochem.

3. Hold at 37oC for ~120 minutes (until % budded cells reaches 95-98%). Remove two samples for 0 time point, then swirl the flask in ice water for one minute and return to 23oC. I usually collect samples every five minutes for 55 minutes, then collect 65 and 80 minute time points.

4. Collecting samples: Freeze 8 ml of 0.1% Na-azide, 0.2 M EDTA in "50 ml" Oakridge-type plastic screw cap centrifuge tubes (freeze these slanted at -20oC to increase surface area). During the experiment, mix 20 ml culture with a 1/20 volume of 10% Na-azide in a 35 ml Corex tube (on ice), immediately transfer to the tube of frozen EDTA /azide, and shake the tube to chill the sample and melt the frozen EDTA. Spin the cells down in a chilled centrifuge, wash with 1 ml cold water in Eppendorf tubes, and freeze the pellets at -20oC.

5. Extract the DNA by smash and grab method.

6. Restriction digest:

a) We usually resuspend each DNA pellet in 20 microliters of TE, then digest half of each sample with EcoRI (this allows us to run two gels from one experiment). I typically use 1 microliter of EcoRI (at 20,000 units/ml) for each sample. After ~6 hours, the DNA is precipitated. One of the 0 minute time samples is saved at this point as a control for the size of the EcoRI fragment (i.e. don't cut one of the samples with DpnI). The rest of the samples are resuspended in 50 microliters water, and an equal volume of a digest mix containing DpnI, restriction buffer, and BSA is added. After 36 hours, the DNA is precipitated and loaded in a 0.7% agarose gel.

b) After blotting the gel, the blot is sequentially hybridized with a fragment adjacent to ARS1 (early replicating), the R11 (late replicating) fragment, and any other fragments of interest.

Recipes

Supplies

Tips

(责任编辑：泉水)

搜索

热门标签:

Replication timing using transient hemimethylation