Mini and Midi prep

From 2007.igem.org

(Difference between revisions)
 
(2 intermediate revisions not shown)
Line 10: Line 10:
inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge
inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge
with rotor and tubes or bottles for harvesting cells)
with rotor and tubes or bottles for harvesting cells)
 +
■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)
■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)
 +
■ Ice
■ Ice
 +
■ Isopropanol
■ Isopropanol
 +
■ 70% ethanol
■ 70% ethanol
 +
■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)
■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)
-
For QIAGEN Plasmid Mini Kit protocol:
+
 
 +
''For QIAGEN Plasmid Mini Kit protocol:''
 +
 
■ Microcentrifuge
■ Microcentrifuge
 +
■ 1.5 ml or 2 ml microcentrifuge tubes
■ 1.5 ml or 2 ml microcentrifuge tubes
-
For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:
+
 
 +
''For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:''
 +
 
■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the
■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the
appropriate protocol.
appropriate protocol.
 +
■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate
■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate
centrifuge tubes or bottles
centrifuge tubes or bottles
Line 26: Line 37:
[[Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit]]
[[Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit]]
-
This protocol is designed for preparation of up to 20 μg of high-copy plasmid or cosmid
+
[[Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits]]
-
DNA using the QIAGEN Plasmid Mini Kit. For additional protocols, such as for cosmid,
+
-
low-copy-number plasmid, BACs, PACs, P1s, and double-stranded M13 replicative form
+
-
purification, see the recommendations at www.qiagen.com/goto/plasmidinfo .
+
-
 
+
-
''Important notes before starting''
+
-
 
+
-
■ New users are advised to familiarize themselves with the detailed protocol provided
+
-
in this handbook. In addition, extensive background information is provided on our
+
-
plasmid resource page www.qiagen.com/goto/plasmidinfo .
+
-
■ Optional: Remove samples at the steps indicated with the symbol ☞ in order to
+
-
monitor the procedure on an analytical gel (see page 41)
+
-
Things to do before starting
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-
■ Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase
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-
A (centrifuge briefly before use) per bottle of Buffer P1, to give a 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.
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-
■ Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use
+
-
one vial LyseBlue (centrifuge briefly before use) per bottle of Buffer P1 to achieve a
+
-
1:1000 dilution. 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.
+
-
 
+
-
''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 approximately 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 3 ml selective LB medium. 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.
+
-
ƒ If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
+
-
4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1.
+
-
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 0.3 ml of 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 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously
+
-
inverting 4–6 times, and incubate on ice for 5 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
+
-
sulphate 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 maximum speed in a microcentrifuge for 10 min. Remove supernatant
+
-
containing plasmid DNA promptly.
+
-
Before loading the centrifuge, the sample should be mixed again. Centrifugation
+
-
should be performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes
+
-
(e.g., 10,000–13,000 rpm in a microcentrifuge). Maximum speed corresponds to
+
-
14,000–18,000 x g for most microcentrifuges. After centrifugation, the supernatant
+
-
should be clear. If the supernatant is not clear, a second, shorter centrifugation should
+
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be carried out to avoid applying any suspended or particulate material to the column.
+
-
Suspended material (which causes the sample to appear turbid) will clog the column
+
-
and reduce or eliminate flow.
+
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☞ Remove a 50 μl sample from the cleared lysate and save it for an analytical gel
+
-
(sample 1).
+
-
8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to
+
-
empty by gravity flow.
+
-
Place QIAGEN-tips into a QIArack over the waste tray or use the tip holders provided
+
-
with each kit (see “Setup of QIAGEN-tips” page 13). 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.
+
-
9. Apply the supernatant from step 7 to the QIAGEN-tip 20 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 before loading to prevent clogging of the QIAGEN-tip.
+
-
☞ Remove a 50 μl sample of the flow-through and save for an analytical gel
+
-
(sample 2).
+
-
10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC.
+
-
Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
+
-
☞ Remove a 220 μl sample of the combined wash fractions and save for an
+
-
analytical gel (sample 3).
+
-
11. Elute DNA with 0.8 ml Buffer QF.
+
-
Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tubes (not supplied).
+
-
Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C
+
-
may help to increase yield.
+
-
☞ Remove a 45 μl sample of the eluate and save for an analytical gel (sample 4).
+
-
12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of
+
-
room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately
+
-
at ≥10,000 rpm for 30 min in a microcentrifuge. Carefully decant the supernatant.
+
-
All solutions should be at room temperature in order to minimize salt precipitation.
+
-
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 easily located.
+
-
Isopropanol pellets are also more loosely attached to the side of the tube, and care
+
-
should be taken when removing the supernatant.
+
-
13. Wash DNA pellet with 1 ml of 70% ethanol and centrifuge at 10,000 rpm for 10 min.
+
-
Carefully decant the supernatant without disturbing the pellet.
+
-
The 70% ethanol removes precipitated salt and replaces isopropanol with the more
+
-
volatile ethanol, making the DNA easier to redissolve.
+
-
14. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer
+
-
(e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH 8.5)
+
-
Redissolve the DNA pellet by rinsing the walls to recover all the DNA. 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.
+
-
Agarose gel analysis
+
-
We recommend removing and saving aliquots during the purification procedure
+
-
(samples 1–4). If the plasmid DNA is of low yield or quality, the samples can be
+
-
analyzed by agarose gel electrophoresis to determine at what stage of the purification
+
-
procedure the problem occurred
+
-
 
+
-
 
+
-
 
+
-
 
+
-
[[Protocol: Plasmid or Cosmid DNA Purification Using
+
-
QIAGEN Plasmid Midi and Maxi Kits]]
+
-
 
+
-
''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 or ● 100 ml 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.
+
-
ƒ 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 30 min 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.
+
-
☞ Remove a ▲ 240 μl or ● 120 μl sample from the cleared lysate supernatant and
+
-
save for an analytical gel (sample 1) in order 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.
+
-
☞ Remove a ▲ 240 μl or ● 120 μl sample from the flow-through and save for an
+
-
analytical gel (sample 2) in order 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 all 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.
+
-
☞ 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.
+
-
☞ Remove a ▲ 100 μl or ● 60 μl sample of the eluate and save for an analytical
+
-
gel (sample 4).
+
-
ƒ If you wish to stop the protocol and continue later, store the eluate at 4°C. Storage
+
-
periods longer than overnight are not recommended.
+
-
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 in order 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 5–10 min, 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 all 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.
+
-
Agarose gel analysis
+
-
We recommend removing and saving aliquots during the purification procedure
+
-
(samples 1–4). If the plasmid DNA is of low yield or quality, the samples can be
+
-
analyzed by agarose gel electrophoresis to determine at what stage of the purification
+
-
procedure the problem occurred
+

Latest revision as of 15:45, 20 September 2007

Equipment and Reagents to Be Supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

For all protocols:

■ Standard microbiological equipment for growing and harvesting bacteria (e.g., inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge with rotor and tubes or bottles for harvesting cells)

■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)

■ Ice

■ Isopropanol

■ 70% ethanol

■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)

For QIAGEN Plasmid Mini Kit protocol:

■ Microcentrifuge

■ 1.5 ml or 2 ml microcentrifuge tubes

For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:

■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the appropriate protocol.

■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate centrifuge tubes or bottles

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits

Retrieved from "http://2007.igem.org/wiki/index.php/Mini_and_Midi_prep"