丁玉强－形态学.pptVIP

8. Resuspend the pellet in 50μl of an RNAse-free solution of 40mM NaHCO3 / 60mM Na2CO3. Remove a 1μl sample for OD260 measurement. Incubate at 60°C for 35 minutes to hydrolyse the probe into small fragments (between 200-300 bp). 35 minutes works fine for a 1 kb probe. ? The amount of time, t , for hydrolysis in 40mM NaHCO3 / 60mM Na2CO3 at 60°C is given by: (Starting length, kb) - (Desired length, kb) t = ————————————————————— (0.11) (Starting length, kb) (Desired length, kb) 9. Purify the probes with Sephadex G-50. In situ manual Principles of in situ procedures All reagents and mailers must be RNAse-free during pre-hybridization Treat all containers (forceps) with DEPC water Wear gloves always Again, please exactly follow the protocols TO ALL FRESHMAN!!!!!!!!!!!! Sections Tissues: Fresh tissue, fixed tissue or paraffin-embedded Sections: 5-20 ?m, collected on Superfrost/Plus slides (Fisher) and dried in air for 1 hour at RT, stored at -20°C (for a long period, at 70°C). Alternatively, use RNAse-free slides coated with TESTA. Details of how to prepare TESTA coated slides are in the Appendix A: Pre-Treatment of Sections 1. Warm slides to RT and dry at 50°C for 15 min. Mark the slide with pencil (probe, date and so on) 2. Fix in 4% PFA in DEPC-PBS at RT for 20 min. 3. Wash twice in DEPC-PBS at RT for 5 min. 4. Treat slides with 50μg/ml Proteinase K in PK buffer at RT for between 8-15 min depending on the age of the embryo. Determine yourself---Concentration or digestion time 5. Wash once in DEPC-PBS at RT for 5 min. 6. Fix in 4% PFA in DEPC-PBS for 15 min 7. Rinse once in DEPC-water 8. Acetylation. Place slides in an RNAse-free glass trough with a stir bar. Add 250ml 0.1M RNAse-free triethanolamine(三乙醇胺)-HCl pH 8.0. Add 0.625ml acetic anhydride with constant stirring. Turn off stirrer when the acetic anhydride (醋酸酐) is dispersed and leave for a further 10 min. Note: Most probes like this, but some do not 9.