提质粒的方法
Qiagen plasmid purification kit
Things to do before starting
_ Add the provided RNase A solutionto Buffer P1 before use. Use 1 vial RNase A
(centrifuge briefly before use) per bottle Buffer P1 for finalconcentration of 100 μg/ml.
_ Check Buffer P2 for SDSprecipitation 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 andmix before use. Use
1 vial LyseBlue reagent per bottle Buffer P1 for a final dilutionof 1:1000 (e.g., 10 μl
LyseBlue into 10 ml Buffer P1). LyseBlue provides visualidentification of optimum
buffer mixing thereby preventing the common handling errors thatlead to inefficient
cell lysis and incomplete precipitation of SDS, genomic DNA, andcell debris. For
more details see “Using LyseBlue reagent” on page 14.
Procedure
1. Pick a single colony from a freshly streakedselective plate and inoculate a starter
culture of 2–5 ml LB medium containing theappropriate 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 volumeof the culture.
2. Dilute the starter culture 1/500 to 1/1000 intoselective 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. 300rpm).
Use a flask or vessel with a volume of at least 4 times the volumeof the culture. The
culture should reach a cell density of approximately 3–4 x 109 cellsper milliliter,
which typically corresponds to a pellet wet weight ofapproximately 3 g/liter
medium.
3. Harvest the bacterial cells by centrifugation at6000 x g for 15 min at 4°C.
If you wish to stop the protocol and continue later, freeze thecell 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 largeenough to allow complete
mixing of the lysis buffers. Ensure that RNase A has been added toBuffer P1.
If LyseBlue reagent has been added to Buffer P1, vigorously shakethe buffer bottle
before use to ensure LyseBlue particles are completelyresuspended. The bacteria
should be resuspended completely by vortexing or pipetting up anddown until no
cell clumps remain.
5. Add_4 ml or _10 ml Buffer P2, mix thoroughly by vigorously inverting thesealed 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. Thelysate should
appear viscous. Do not allow the lysis reaction to proceed formore than 5 min. After
use, the bottle containing Buffer P2 should be closed immediatelyto avoid
acidification from CO2 in the air.
If LyseBlue has been added to Buffer P1, the cell suspension willturn blue after
addition of Buffer P2. Mixing should result in a homogeneouslycolored suspension.
If the suspension contains localized colorless regions or ifbrownish cell clumps are
still visible, continue mixing the solution until a homogeneouslycolored suspension
is achieved.
6. Add _4 ml or _10 ml of chilled Buffer P3, mix immediately and thoroughlyby
vigorously inverting 4–6 times, and incubate on icefor _ 15 min or _ 20 min.
Precipitation is enhanced by using chilled Buffer P3 andincubating on ice. After
addition of Buffer P3, a fluffy white material forms and thelysate becomes less
viscous. The precipitated material contains genomic DNA, proteins,cell debris, and
KDS. The lysate should be mixed thoroughly to ensure evenpotassium dodecyl sulfate
precipitation. If the mixture still appears viscous, more mixingis required to
completely neutralize the solution.
If LyseBlue reagent has been used, the suspension should be mixeduntil all trace of
blue has gone and the suspension is colorless. A homogeneouscolorless suspension
indicates that the SDS has been effectively precipitated.
7. Centrifuge at ≥20,000 x g for 30min at 4°C. Removesupernatant 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 beefficiently cleared by
filtration using a QIAfilter Kits or Cartridges (seewww.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 avoidapplying suspended
or particulate material to the QIAGEN-tip. Suspended material(causing the sample
to appear turbid) can clog the QIAGEN-tip and reduce or eliminategravity flow.
Optional: Remove a _ 240 μl or _ 120 μl sample from the cleared lysate
supernatant and save for an analytical gel (sample 1) to determinewhether 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 gravityflow.
Flow of buffer will begin automatically by reduction in surfacetension due to the
presence of detergent in the equilibration buffer. Allow theQIAGEN-tip to drain
completely. QIAGEN-tips can be left unattended, since the flow ofbuffer will stop
when the meniscus reaches the upper frit in the column.
10. Apply the supernatant from step 8 to theQIAGEN-tip and allow it to enter the resin
by gravity flow.
The supernatant should be loaded onto the QIAGEN-tip promptly. Ifit is left too long
and becomes cloudy due to further precipitation of protein, itmust be centrifuged
again or filtered before loading to prevent clogging of theQIAGEN-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 ofDNA 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 DNApreparations. The
second wash is especially necessary when large culture volumes orbacterial 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 ofpolycarbonate
centrifuge tubes is not recommended as polycarbonate is notresistant to the alcohol
used in subsequent steps.
Note: For constructs larger than 45–50 kb, prewarming the elutionbuffer 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 theeluate 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 centrifugeimmediately at ≥15,000 x g for
30 min at 4°C. Carefully decant the supernatant.
All solutions should be at room temperature to minimize saltprecipitation, although
centrifugation is carried out at 4°C to prevent overheating of thesample.
Alternatively, disposable conical bottom centrifuge tubes can beused for
centrifugation at 5000 x g for 60 min at 4°C. Isopropanolpellets 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 thetube 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 betaken 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 thesupernatant without
disturbing the pellet.
Alternatively, disposable conical-bottom centrifuge tubes can beused 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 redissolvethe 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 promoteresuspension may
cause shearing and should be avoided. Overdrying the pellet willmake the DNA
difficult to redissolve. DNA dissolves best under slightlyalkaline conditions; it does
not easily dissolve in acidic buffers.
Determination of yield
To determine the yield, DNA concentration should be determined byboth UV
spectrophotometry at 260 nm and quantitative analysis on anagarose gel. For reliable
spectrophotometricDNA quantification, A260 readings should lie between 0.1 and 1.0.