提质粒的方法

Qiagen plasmid purification kit

 

Things to do before starting

_ Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A

(centrifuge briefly before use) per bottle Buffer P1 for final concentration of 100 μg/ml.

_ Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary,

dissolve the SDS by warming to 37°C.

_ Pre-chill Buffer P3 at 4°C.

_ Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use

1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 μl

LyseBlue into 10 ml Buffer P1). LyseBlue provides visual identification of optimum

buffer mixing thereby preventing the common handling errors that lead to inefficient

cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. For

more details see “Using LyseBlue reagent” on page 14.

 

Procedure

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter

culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate

for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).

Use a tube or flask with a volume of at least 4 times the volume of the culture.

 

2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy

plasmids, inoculate _ 25 ml (midi extract)or _ 100 ml (Maxi extract) medium with _ 25–50 μl or

_ 100–200 μl of starter culture. For low-copy plasmids, inoculate _ 100 ml or

_ 500 ml medium with _ 100–200 μl or _ 250–500 μl of starter culture. Grow at

37°C for 12–16 h with vigorous shaking (approx. 300 rpm).

Use a flask or vessel with a volume of at least 4 times the volume of the culture. The

culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,

which typically corresponds to a pellet wet weight of approximately 3 g/liter

medium.

3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.

If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.

4. Resuspend the bacterial pellet in _ 4 ml or _ 10 ml Buffer P1.

For efficient lysis, it is important to use a vessel that is large enough to allow complete

mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.

If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle

before use to ensure LyseBlue particles are completely resuspended. The bacteria

should be resuspended completely by vortexing or pipetting up and down until no

cell clumps remain.

5. Add_ 4 ml or _ 10 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.

Do not vortex, as this will result in shearing of genomic DNA. The lysate should

appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After

use, the bottle containing Buffer P2 should be closed immediately to avoid

acidification from CO2 in the air.

If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after

addition of Buffer P2. Mixing should result in a homogeneously colored suspension.

If the suspension contains localized colorless regions or if brownish cell clumps are

still visible, continue mixing the solution until a homogeneously colored suspension

is achieved.

6. Add _ 4 ml or _ 10 ml of chilled Buffer P3, mix immediately and thoroughly by

vigorously inverting 4–6 times, and incubate on ice for _ 15 min or _ 20 min.

Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After

addition of Buffer P3, a fluffy white material forms and the lysate becomes less

viscous. The precipitated material contains genomic DNA, proteins, cell debris, and

KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate

precipitation. If the mixture still appears viscous, more mixing is required to

completely neutralize the solution.

If LyseBlue reagent has been used, the suspension should be mixed until all trace of

blue has gone and the suspension is colorless. A homogeneous colorless suspension

indicates that the SDS has been effectively precipitated.

7. Centrifuge at ≥20,000 x g for 30min at 4°C. Remove supernatant containing plasmid

DNA promptly.

Before loading the centrifuge, the sample should be mixed again. Centrifugation

should be performed in non-glass tubes (e.g., polypropylene). After centrifugation

the supernatant should be clear.

Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by

filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/

plasmid/LargeScaleKits).

8. Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove

supernatant containing plasmid DNA promptly.

This second centrifugation step should be carried out to avoid applying suspended

or particulate material to the QIAGEN-tip. Suspended material (causing the sample

to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.

Optional: Remove a _ 240 μl or _ 120 μl sample from the cleared lysate

supernatant and save for an analytical gel (sample 1) to determine whether growth

and lysis conditions were optimal.

9. Equilibrate a _ QIAGEN-tip 100 or _ QIAGEN-tip 500 by applying _ 4 ml or

_ 10 ml Buffer QBT, and allow the column to empty by gravity flow.

Flow of buffer will begin automatically by reduction in surface tension due to the

presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain

completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop

when the meniscus reaches the upper frit in the column.

10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin

by gravity flow.

The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long

and becomes cloudy due to further precipitation of protein, it must be centrifuged

again or filtered before loading to prevent clogging of the QIAGEN-tip.

Optional: Remove a _ 240 μl or _ 120 μl sample from the flow-through and save

for an analytical gel (sample 2) to determine the efficiency of DNA binding to the

QIAGEN resin.

11. Wash the QIAGEN-tip with _ 2 x 10 ml or _ 2 x 30 ml Buffer QC.

Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is

sufficient to remove contaminants in the majority of plasmid DNA preparations. The

second wash is especially necessary when large culture volumes or bacterial strains

producing large amounts of carbohydrates are used.

Optional: Remove a_ 400 μl or _ 240 μl sample from the combined wash fractions

and save for an analytical gel (sample 3).

12. Elute DNA with _ 5 ml or _ 15 ml Buffer QF.

Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate

centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol

used in subsequent steps.

Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C

may help to increase yield.

Optional: Remove a _ 100 μl or _ 60 μl sample of the eluate and save for an

analytical gel (sample 4).

If you wish to stop the protocol and continue later, store the eluate at 4°C. Storage

periods longer than overnight are not recommended.mid 

Midi andMaxi Kits

13. Precipitate DNA by adding _ 3.5 ml or _ 10.5 ml (0.7 volumes) room-temperature

isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for

30 min at 4°C. Carefully decant the supernatant.

All solutions should be at room temperature to minimize salt precipitation, although

centrifugation is carried out at 4°C to prevent overheating of the sample.

Alternatively, disposable conical bottom centrifuge tubes can be used for

centrifugation at 5000 x g for 60 min at 4°C. Isopropanol pellets have a glassy

appearance and may be more difficult to see than the fluffy, salt-containing pellets

that result from ethanol precipitation. Marking the outside of the tube before

centrifugation allows the pellet to be more easily located. Isopropanol pellets are also

more loosely attached to the side of the tube, and care should be taken when

removing the supernatant.

14. Wash DNA pellet with _ 2 ml or _ 5 ml of room-temperature 70% ethanol, and

centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without

disturbing the pellet.

Alternatively, disposable conical-bottom centrifuge tubes can be used for

centrifugation at 5000 x g for 60 min at 4°C. The 70% ethanol removes precipitated

salt and replaces isopropanol with the more volatile ethanol, making the DNA easier

to redissolve.

15. Air-dry the pellet for 5–10min, and redissolve the DNA in a suitable volume of buffer

(e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

Redissolve the DNA pellet by rinsing the walls to recover the DNA, especially if glass

tubes have been used. Pipetting the DNA up and down to promote resuspension may

cause shearing and should be avoided. Overdrying the pellet will make the DNA

difficult to redissolve. DNA dissolves best under slightly alkaline conditions; it does

not easily dissolve in acidic buffers.

Determination of yield

To determine the yield, DNA concentration should be determined by both UV

spectrophotometry at 260 nm and quantitative analysis on an agarose gel. For reliable

spectrophotometric DNA quantification, A260 readings should lie between 0.1 and 1.0.