实验试剂

1. Absolute ethanol (96%-100%) - Do not use other alcohols

2. 2-mercaptoethanol

实验设备

1. Tabletop micro-centrifuge and nuclease-free 1.5 ml tubes

2. Water bath set to 30°C

实验步骤

1. Inoculate 5 ml YPD medium placed in a 10-20 ml culture tube with Yeast carrying desired plasmid and grow at 30°C with agitation for 20-24 h.

2. Pellet 1-3 ml yeast culture (use < 2 x 107 cells) by centrifugation at 4,000×g for 5 min at room temperature.

3. Discard medium and resuspend cells in 480 ul Buffer SE, 10 ul 2-mercaptoethanol and 20 ul lyticase solution. Resuspend the pellet by vortexing or pipetting. Complete resuspension of cell pellet is vital of obtaining good yields. Incubate at 30°C for at least 30 min.

4. Pellet spheroblasts by centrifuging at 4,000 x g for 5 min at room temperature. Discard the supernatant completely.

5. Resuspend the spheroblasts pellet with 250 ul Buffer YP I/RNase A.

6. Add 50mg glass beads and vortex at maxi speed for 5 minutes. Let it stand to allow the beads to settle. Transfer the supernatant to a new 1.5 ml centrifuge tube (not supplied).

7. Add 250 ul YP II and gently mix by inverting and rotating tube 4-6 times to obtain a cleared lysate. A 2 min incubation at room temperature may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. (Store YP II tightly capped when not in use.)

8. Add 350 ul YP III and gently mix by inverting several times until a flocculent white precipitate forms. Centrifuge at $10,000 × g for 10 minutes at room temperature.

9. CAREFULLY aspirate and add the clear supernatant to a clean HiBind® DNA mini column assembled in a 2 ml collection tube (provided). Ensure that the pellet is not disturbed and that no cellular debris is carried over into the column. Centrifuge for 1 min at 10,000 × g at room temperature to completely pass lysate through column.

10. Discard flow-through liquid and wash the column by adding 500 ul of Buffer HB. Centrifuge for 1 min at 10,000 × g as above.

11. Discard flow-through liquid and wash the column by adding 700 ul of DNA Wash Buffer diluted with ethanol. Centrifuge for 1 min at 10,000 × g as above and discard flow-through.

NOTE: DNA Wash Buffer is supplied as a concentrate and must be diluted with absolute ethanol according to the instructions on bottle or on Page 3 under “Before Starting.”

12. Optional Step: Repeat wash step with another 700 ul DNA Wash Buffer.

13. Centrifuge the empty column for 2 min at $13,000 × g to dry the column matrix. Do not skip this step - it is critical for removing ethanol from the column.

14. Place column into a clean 1.5 ml microcentrifuge tube. Add 30-50 ul (depending on desired concentration of final product) sterile deionized water (or TE buffer) directly onto the column matrix and centrifuge for 1 min at 10,000 ×g to elute DNA. This represents approximately 75-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

15. Yield and quality of DNA: Determine the absorbance of an appropriate dilution (20- to 50-fold) of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows:

DNA concentration = Absorbance260 × 50 × (Dilution Factor) :g/ml

Although the binding capacity of HiBind® DNA Column is around 35 ug, the yield of the yeast plasmid depends on the yeast strain and type of plasmid. High copy number plasmids generally yield up to 1ug of DNA from 5 ml culture. The ratio of (absorbance260)/(absorbance280) is an indication of nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid. Alternatively, quantity (as well as quality) can sometimes best be determined by agarose gel/ethidium bromide electrophoresis by comparison to DNA samples of known concentrations. Typically, the majority of the DNA eluted is in monomeric supercoil form, though concatamers may also be present.