相关专题  

问：我欲提酵母质粒 。我在为酵母细胞破碎犯愁，有人说超声波就可以。但大多人说对于酵母不行，高手们请指教了!谢了!

答：军科院的方法：

1、用酵母细胞裂解液重悬酵母菌

2、加酚-氯仿

3、加玻璃珠

4、振荡半小时

5、乙醇沉淀

6、电转大肠 杆菌

Detailed:

Plasmid Isolation from Yeast

Reagents and Materials Required

The YEASTMAKER Yeast Plasmid Isolation Kit (#K1611-1) provides the SDS and lyticase solutions, CHROMA SPIN-

1000 DEPC-H2O Columns, and 2-ml centrifuge tubes for use with the columns.

• Appropriate SD liquid or agar medium to keep selection on the plasmids (Appendix C.A;

Appendix E).

• Sterile, 1.5-ml microcentrifuge tubes (or a 96-tube microtiter array, multichannel pipettors, and

centrifuge adaptor for multiwell plates).

• 20% SDS

• Lyticase Solution (5 units/ml in TE buffer; store at 4°C for up to 2 months or at –20°C for up to

6 months. If colloidal material precipitates, mix the solution by inversion before using.)

• Recommended: CHROMA SPIN-1000 DEPC-H2O Columns (#K1334-1) and 2-ml centrifuge

tubes for use with the columns

• If you do not use CHROMA SPIN Columns, you will need materials to perform phenol:chloroform

extraction and ethanol precipitation:

• Phenol:chloroform:isoamyl alcohol (25:24:1; See Sambrook et al., 1989, for information on

preparing neutralized phenol solutions)

• 10 M ammonium acetate

• 95–100% ethanol

1.Prepare yeast cultures for lysis (Step a, b, or c below).

a. From a solid patch of growth:

i. Spread a thin film of yeast cells (~2-cm2 patch) onto the appropriate SD agar medium.

ii. Incubate plate at 30°C for 3–4 days. (The patch should show abundant yeast growth.)

iii. Scrape up a portion of the patch (~10 mm2) and resuspend the cells in 50 ml of sterile

H2O or TE in a 1.5-ml microcentrifuge tube.

b. From a liquid culture:

i. Inoculate a large (2–4-mm), fresh (2–4-day-old) yeast colony into 0.5 ml of the

appropriate SD liquid medium. Vortex tube vigorously to completely break up the colony

and resuspend the cells.

ii. Incubate at 30°C overnight with shaking at 230–250 rpm.

iii. Spin down the cells by centrifuging at 14,000 rpm for 5 min.

iv. Carefully pour off the supernatant and resuspend pellets in the residual liquid (total

volume ~50 ml).

c. For semi-automated handling of a large number of samples:

i. Place a large (2–4-mm), fresh (2–4-day-old) yeast colony into 0.5 ml of the appropriate

SD liquid medium in separate wells of a 96-tube microtiter array. Vortex each tube

vigorously to resuspend the cells. (Alternatively, use 0.5 ml of an overnight SD liquid

culture instead of a yeast colony.)

ii. Using a centrifuge adapted for multiwell plates, centrifuge the entire array at 1,000 x g

for 5 min to pellet the cells.

iii. Carefully pour (or draw) off supernatants and resuspend pellets in the residual medium

(~50 ml) by vortexing or pipetting up and down.

2. Add 10 ml of lyticase solution to each tube. Thoroughly resuspend the cells by vortexing

or repeatedly pipetting up and down.

3. Incubate tubes at 37°C for 30–60 min with shaking at 200-250 rpm.

[Optional] Check a drop of the cell suspension under a phase contrast microscope (400X) for the progress of

cell lysis by adding a drop of 20% SDS to the side of the coverslip. As they come into contact with the SDS, most

cells should lose their refractile appearance and appear as "ghost-like" spheroplasts. If there are still many intact

cells present, incubate the samples for another 30 min.

4. Add 10 ml of 20% SDS to each tube and vortex vigorously for 1 min to mix.

5. Put the samples through one freeze/thaw cycle (at –20°C) and vortex again to ensure

complete lysis of the cells.

6. If necessary, samples can be stored frozen at –20°C. If samples have been frozen, vortex

them again before using them.

7. Pour the entire contents of the tube from Step 5 above onto a prespun CHROMA SPIN-

1000 Column and purify the plasmid DNA according to the CHROMA SPIN User Manual.

Purified plasmid DNA will elute from the column.

If you do not use CHROMA SPIN Columns, clean up the prep as follows:

a. Bring the volume of the sample up to 200 ml in TE buffer (pH 7.0).

b. Add 200 ml of phenol:chloroform:isoamyl alcohol (25:24:1).

c. Vortex at highest speed for 5 min.

d. Centrifuge at 14,000 rpm for 10 min.

e. Transfer the aqueous (upper) phase to a fresh tube.

f. Add 8 ml of 10 M ammonium acetate and 500 ml of 95–100% Ethanol.

h. Place at –70°C or in a dry-ice/ethanol bath for 1 hr.

i. Centrifuge at 14,000 rpm for 10 min.

j. Discard supernatant and dry the pellet.

k. Resuspend pellet in 20 ml of H2O.

Note: The amount of plasmid DNA recovered is small relative to the contaminating genomic DNA;

therefore, it cannot be measured by A260 or seen on an agarose gel.