酵母单杂交系统与实验操作.pptVIP

B. Good Laboratory Practices When resuspending pellets or mixing reactions, gently pipet the solution up and down or tap the bottom of the tube. Spin briefly to bring contents to the bottom of the tube. Do not vortex samples when resuspending pellets; vortexing may shear your cDNA. Perform all reactions on ice, unless otherwise indicated. Do not increase the size (volume) of any of the reactions. All components have been optimized for the volumes specified. In preparing your reactions, use the Deionized H2O supplied. IV.Generating a cDNA Library C.RNA Isolation The minimum amount of starting material for cDNA synthesis is 100 ng of total RNA or 25 ng of poly A+ RNA.Use the higher starting amounts of RNA shown in the table. D. RNA Analysis IV.Generating a cDNA Library 图5 拟南芥叶片总RNA的提取 Figure. 5 Isolation of total RNA from Arabidopsis 18S 28S E. Synthesize First-Strand cDNA using an Oligo (dT) Primer 1. Combine the following reagents in a sterile 0.25-ml microcentrifuge tube:1–2 μl RNA sample (0.025–1.0 μg poly A+ or 0.10–2.0 μg total RNA),1.0 μl CDS III Primer,1–2 μl Deionized H2O to bring volume up to 4.0 μl.,4.0 μl Total volume 2. Mix contents and spin briefly. 3. Incubate at 72°C for 2 min. 4. Cool on ice for 2 min. 5. Spin briefly. 6. Add the following to the reaction tube:2.0 μl 5X First-Strand Buffer,1.0 μl DTT (20 mM),1.0 μl dNTP Mix (10 mM ),1.0 μl MMLV Reverse Transcriptase,9.0 μl Total volume 7. Mix gently by tapping. Spin briefly. 8. Incubate at 42°C for 10 min. 9. Add 1.0 μl BD SMART III Oligonucleotide. 10. Incubate at 42°C for 1 hr in an air incubator or hot-lid thermal cycler. 11. Place the tube at 75°C for 10 min to terminate first-strand synthesis. 12. Cool the tube to room temperature, then add 1.0 μl (2 units) RNase H. 13. Incubate at 37°C for 20 min. 14. If you plan to proceed directly to the LD-PCR step, take a 2-μl aliquot from the first-strand synthesis and place it in a clean, prechilled, 0.5-ml tube. Pla