GeneJET_Plasmid_Midiprep_kit
GeneJET_Plasmid_Midiprep_kit
GeneJET_Plasmid_Midiprep_kit
Thermo Scientific
GeneJET Plasmid Midiprep Kit
#K0481, #K0482
www.thermoscientific.com/onebio
#K0481, #K0482
Lot ___
Expiry Date _
CERTIFICATE OF ANALYSIS
Thermo Scientific GeneJET Plasmid Midiprep Kit is qualified by isolating high copy plasmid DNA
from 50 mL of overnight E. coli culture (OD600 = 2) grown in LB medium following the protocol
outlined in the manual. The kit passes QC requirements if the purified plasmid DNA has an
A260/A280 ratio between 1.8 and 1.9 and a dominant band of supercoiled plasmid DNA is observed
following agarose gel electrophoresis. The functional quality of the plasmid DNA is evaluated by
digestion with restriction endonucleases.
Rev.7.
CONTENTS
DESCRIPTION ............................................................................................................................... 2
PRINCIPLE .................................................................................................................................... 2
PROTOCOLS ................................................................................................................................. 5
TROUBLESHOOTING ................................................................................................................... 8
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COMPONENTS OF THE KIT
#K0481 #K0482
GeneJET Plasmid Midiprep Kit
25 preps 100 preps
Resuspension Solution 60 mL 240 mL
Lysis Solution 60 mL 240 mL
Neutralization Solution 60 mL 240 mL
Endotoxin Binding Reagent 15 mL 60 mL
Wash Solution I (concentrated) 90 mL 360 mL
Wash Solution II (concentrated) 90 mL 2 × 180 mL
RNase A Solution 2 × 1.2 mL 8 × 1.2 mL
Elution Buffer (10 mM Tris-HCl, pH 8.5) 18 mL 60 mL
GeneJET Midi Purification Columns pre-assembled
25 100
with collection tubes (15 mL)
Collection tubes (15 mL) 25 100
DESCRIPTION
The GeneJET™ Plasmid Midiprep Kit is designed for large scale isolation of high quality plasmid DNA
from recombinant E.coli cultures. The kit utilizes silica-based membrane technology in the form of
convenient spin columns. Each prep recovers up to 200 µg of high copy number plasmid DNA. The
purified plasmid DNA can be used in a wide variety of molecular biology procedures such as restriction
endonuclease digestion, PCR, cloning, transformation, automated sequencing, in vitro transcription and
transfection of robust cell lines.
PRINCIPLE
Pelleted bacterial cells are resuspended and subjected to SDS/alkaline lysis to liberate plasmid
DNA. The resulting lysate is neutralized allowing denatured plasmid DNA to reanneal while cell
debris such as proteins, chromosomal DNA and SDS precipitate out of solution. The resulting
precipitate is pelleted by centrifugation and the supernatant containing plasmid DNA is loaded
onto the purification column. The high salt concentration of the lysate creates the appropriate
conditions for plasmid DNA binding to the silica membrane in the spin column. The adsorbed DNA
is washed to remove contaminants and eluted with the Elution Buffer.
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IMPORTANT NOTES
Buffer preparation and handling
• Add the RNase A Solution included in the kit to the Resuspension Solution and mix
thoroughly. After the addition of RNase A, the Resuspension Solution is stable for 6 months
when stored at 4°C.
• If the kit will be used infrequently, divide the Resuspension Solution into an appropriate number
of aliquots and supplement one aliquot with 40 µL of RNase A per 1 mL of Resuspension
Solution to create a working solution. Store the remaining RNase A at -20°C. Sequential
working solutions can be prepared by supplementing an aliquot of the Resuspension Solution
with RNase A.
• Add the indicated volume of isopropanol to Wash Solution I (concentrated) and ethanol (96-
100%) to Wash Solution II (concentrated) prior to first use:
• Check the Lysis Solution and the Neutralization Solution for salt precipitation before each
use. Re-dissolve any precipitate by warming the solution at 37°C, then cool back down to
25°C before use. Do not vigorously shake the Lysis Solution.
• Wear gloves when handling the Lysis Solution and Wash Solution I bottles as these
solutions contain irritants (see p.12 for SAFETY INFORMATION) and are harmful if they come
in contact with skin, or if they are inhaled or swallowed.
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Bacterial strains
High quality plasmid DNA can be obtained from various E.coli strains including DH10B, DH5α,
XL1-Blue, JM109, JM107, TOP10.
Culture media
The GeneJET Plasmid Midiprep purification protocol outlined in this manual is optimized for
use with bacterial cultures grown in Luria-Bertani (LB) medium to a optical density of
approximately 2-3 (OD600 = 2-3). The use of rich growth media is not recommended.
Culture volume
Generally, a 50 mL of overnight bacterial culture grown in LB medium is sufficient for good
yield of high-copy and low-copy number plasmid DNA. However, it is important not to exceed
recommended cell mass (culture volume × OD600), because it may decrease quality of isolated
DNA. The maximum culture volume to use can be determined using formula below:
Maximum culture volume (mL) = 150/OD600
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PROTOCOLS
The GeneJET Plasmid Midi Prep Kit provides optimized protocols for plasmid DNA purification
using low speed (up to 5,000 × g) and high speed (up to 48,000 × g) centrifuges, as well as using
vacuum manifolds.
Note
• Harvest the bacterial culture by centrifugation at +4°C. All others centrifugation steps should be
carried out at room temperature.
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column. Centrifuge for 2 min. at 3,000 × g in a swinging bucket rotor. Discard the flow-through and
place the column back into the same collection tube.
13 Repeat the column wash with Wash Solution II (step 12).
Centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to remove residual wash solution.
14
Discard the collection tube containing the flow-through.
Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of the Elution
Buffer to the centre of the purification column membrane. Incubate for 2 min at room temperature
and centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to elute plasmid DNA.
Note. To increase the concentration of eluted DNA the volume of the Elution Buffer can be
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reduced to 0.25 mL. Be aware that lower volumes of Elution Buffer will decrease the overall yield
of eluted DNA.
To increase the overall DNA yield by 20-30% an additional elution step (optional) with Elution
Buffer (0.15 mL) may be used.
Discard the purification column. Use the purified plasmid DNA in downstream applications or store
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DNA at -20°C.
Protocol B. Plasmid DNA purification using high speed centrifuges
Step Procedure
Grow up to 50 mL of bacterial culture to an OD600 of 2-3 as outlined on p.4.
For best results calculate the maximum volume of cell culture to use by referring to the
1
“Culture volume” on p.4.
Harvest the cells by centrifugation for 10 min at 5,000 × g. Discard the supernatant.
Resuspend the pelleted cells in 2 mL of Resuspension Solution. The bacterial pellet should be
resuspended by vortexing or pipetting up and down until no cell clumps remain.
2
Note. Ensure that RNase A Solution has been added to the Resuspension Solution as described
on p.3.
Add 2 mL of Lysis Solution and mix gently by inverting the tube 4-6 times until the solution
becomes viscous and slightly clear. Incubate for 3 min at room temperature.
3
Note. Do not vortex to avoid shearing chromosomal DNA. Do not incubate for more than 3 min
to avoid denaturation of supercoiled plasmid DNA.
4 Add 2 mL of the Neutralization Solution and mix immediately by inverting the tube 5-8 times.
Add 0.5 mL of the Endotoxin Binding Reagent. Mix immediately by inverting the tube 5-8 times.
Incubate for 5 min at room temperature.
5 Note. After the addition of the Neutralization Solution and Endotoxin Binding Reagent it is
important to mix gently, but thoroughly, to avoid localized precipitation of bacterial cell debris. The
neutralized bacterial lysate should appear cloudy and contain white precipitate.
Centrifuge for 20 min at 20,000 rpm (48,000 × g) to pellet cell debris and chromosomal DNA.
6 (see p. 9 to continue with vacuum protocol, if preferred).
Note. Use only recommended centrifugation speed.
Transfer the supernatant into a 15 mL tube (not provided) by decanting or pipetting. Avoid
7 disturbing or transferring the white precipitate.
Add 1 volume of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
Transfer part of the sample (~ 5.5 mL) to the supplied column pre-assembled with a collection
tube (15 mL). Be careful not to overfill the column. Centrifuge for 3 min at 2,000 × g in a swinging
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bucket rotor. Discard the flow-through and place the column back into the same collection tube.
Note. Close the bag with columns tightly after use!
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9 Repeat step 8 to process any remaining lysate through the purification column.
Add 4 mL of Wash Solution I (diluted with isopropanol as described on p. 3) to the purification
10 column. Centrifuge for 2 min. at 3,000 × g in a swinging bucket rotor. Discard the flow-through and
place the column back into the same collection tube.
Add 4 mL of Wash Solution II (diluted with ethanol as described on p. 3) to the purification
11 column. Centrifuge for 2 min. at 3,000 × g in a swinging bucket rotor. Discard the flow-through and
place the column back into the same collection tube.
12 Repeat the column wash with Wash Solution II (step 11).
Centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to remove residual wash solution.
13
Discard the collection tube containing the flow-through.
Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of the Elution
Buffer to the centre of the purification column membrane. Incubate for 2 min at room temperature
and centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to elute plasmid DNA.
Note. To increase the concentration of eluted DNA the volume of the Elution Buffer can be
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reduced to 0.25 mL. Be aware that lower volumes of Elution Buffer will decrease the overall yield
of eluted DNA.
To increase the overall DNA yield by 20-30% an additional elution step (optional) with Elution
Buffer (0.15 mL) may be used.
Discard the purification column. Use the purified plasmid DNA in downstream applications or store
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DNA at -20°C.
Protocol C. Plasmid DNA purification using vacuum protocol
Perform cell collection, lysis and lysate clearing steps according steps 1 - 8 in Protocol A on page
1
5 or steps 1 - 7 in Protocol B on page 7.
Prepare the vacuum manifold according to the supplier’s instructions. Place the GeneJET Midi
2
Purification column(s) onto the manifold.
Transfer part of the sample (~ 5.5 mL) to the column. Be careful not to overfill the column. Apply
the vacuum to draw the solution through the column. Switch off the vacuum after the solution has
3
passed through the column.
Note. Close the bag with columns tightly after use!
4 Repeat step 3 to draw the remaining lysate through the purification column.
Add 4 mL of the Wash Solution I (diluted with isopropanol as described on p.3) to the purification
5 column. Apply the vacuum to draw the solution through the column. Switch off the vacuum after
the solution has passed through the column.
Add 4 mL of Wash Solution II (diluted with ethanol as described on p.3) to the purification
6 column. Apply the vacuum to draw the solution through the column. Switch off the vacuum after
the solution has passed through the column.
7 Repeat the column wash with Wash Solution II (step 6).
To dry the column, apply the vacuum for 10 min or transfer the column into a fresh 15 mL
8
collection tube (provided) and centrifuge for 5 min. at 3,000 × g in a swinging bucket rotor.
Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of Elution Buffer
to the centre of the purification column membrane. Incubate for 2 min at room temperature and
9 centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to elute plasmid DNA.
Note. If more concentrated DNA samples are required the volume of Elution Buffer added to the
column membrane can be reduced to 0.25 mL. Please be aware that smaller volumes of the
7
Elution Buffer will decrease the overall yield of the eluted DNA.
To increase the overall DNA yield by 20-30% an additional elution step (optional) with Elution
Buffer (0.15 mL) may be used.
Discard the purification column. Use the purified plasmid DNA for downstream applications or
10
store the DNA at -20°C.
8
TROUBLESHOOTING
Problem Possible cause and solution
Incomplete bacterial cell lysis. It is essential that the cell pellet is completely resuspended in
Resuspension Solution prior to lysis. There should be no visible cell clumps before adding the
Lysis Solution.
Check the Lysis Solution for salt precipitation before each use. Redissolve any precipitate by
warming the solution to 37°C, then mix well and cool to 25°C before use.
Do not use more cell culture biomass than recommended. Calculate the maximum culture
volume to use by referring to the “Culture volume” on p.4.
Isopropanol was not added to Wash Solution I. Ensure that isopropanol was added to
Wash Solution I before the first use. Follow the instructions to prepare Wash Solution I on p.3.
Ethanol was not added to Wash Solution II. Ensure that ethanol was added to Wash
Solution II before the first use. Follow the instructions to prepare Wash Solution II on p.3.
Low plasmid
Column clogs during the purification procedure. Reduce volume of cell culture biomass
DNA yield
processed per column.
Avoid transferring pelleted cell debris to the purification column when loading the lysate.
Suboptimal cell culture volume. Calculate the maximum volume using the following formula:
Maximum culture volume (mL) = 150/OD600.
Old bacterial culture
Prepare new starter culture by inoculating a freshly-isolated single bacterial colony in
antibiotic-containing growth medium and grow bacteria as described in “Bacterial Culture
Growth” on p.3.
Inappropriate centrifuge speed.
Please make sure that recommended centrifuge speed was used during purification. It is very
important to meticulously adhere to the centrifugation protocol.
Suboptimal Inefficient plasmid DNA purification. Reduce cell culture volume. Follow recommendations
A260/A280 ratio described in “Culture volume” on p.4.
Samples vigorously vortexed or shook during cell lysis or neutralization steps. To avoid
genomic DNA contamination, mix the solution (steps 3 and 4) by gently inverting the tube 4-8
times.
Genomic DNA
Do not allow the cell lysis step (step 3) to proceed for more than 3 min.
contamination
Do not cultivate cells longer than 16 h in LB media.
Residual genomic DNA can be removed from purified plasmid DNA by treatment using T7
DNA Polymerase (#EP0081).
RNA RNase A Solution was not added to the Resuspension Solution. Ensure that the RNase A
contamination Solution was added to the Resuspension Solution as described on p.3.
Purified prep
Plasmid DNA denatured during cell lysis. Denatured plasmid DNA migrates ahead of
contains
supercoiled DNA and is not suitable for enzymatic manipulations such as restriction digestion.
additional
To avoid denaturation, do not allow the cell lysis (step 3) to proceed for more than 3 min.
plasmid forms
Inhibition of
downstream Purified plasmid DNA contains ethanol. Dry the purification column sufficiently before the
enzymatic DNA elution (steps 13 or 14 of the centrifugation protocols or step 8 of the vacuum protocol).
reactions
No or low yields
of in vitro Purified plasmid DNA contains RNase A. If the purified DNA is used for in vitro
transcription transcription, linearized plasmid DNA can be repurified using the GeneJET PCR Purification
reaction Kit (#K0701) or by phenol/chloroform extraction.
products
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References
1. H.C. Birnboim, J. Doly, A rapid alkaline lysis procedure for screening recombinant plasmid DNA.
Nucleic Acids Res. 7, 1513 -1522 (1979).
2. B. Vogelstein, D. Gillespie, Preparative and analytical purification of DNA from agarose. Proc. Natl.
Acad. Sci. USA 76, 615-619 (1979).
SAFETY INFORMATION
Lysis Solution
Corrosive
Hazard-determining components of labeling: sodium hydroxide
Risk phrases
R34 Causes burns.
Safety phrases
S20 When using do not eat or drink.
S23 Do not breathe gas/fumes/vapor/spray.
S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
S36/37/39 Wear suitable protective clothing, gloves and eye/face protection.
S45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
S60 This material and its container must be disposed of as hazardous waste.
Lysis Solution
Danger
Hazard statements:
H314 Causes severe skin burns and eye damage.
H319 Causes serious eye irritation.
Precautionary statements:
P260 Do not breathe dust/fume/gas/mist/vapours/spray.
P303+P361+P353 IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy
to do. Continue rinsing.
P310 Immediately call a POISON CENTER or doctor/physician.
P405 Store locked up. P501 Dispose of contents/container in accordance with local/regional/national/ international
regulations.
Neutralization Solution
Xi Irritant
Hazard-determining components of labeling: acetic acid
Risk phrases:
R36/38 Irritating to eyes and skin.
Safety phrases:
23 Do not breathe gas/fumes/vapour/spray.
26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
37 Wear suitable gloves.
45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
60 This material and its container must be disposed of as hazardous waste.
10
Neutralization Solution
Warning
Hazard statements:
H315 Causes skin irritation.
H319 Causes serious eye irritation.
Precautionary statements:
P280 Wear protective gloves/protective clothing/eye protection/face protection.
P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy
to do. Continue rinsing.
P321 Specific treatment (see on this label).
P362 Take off contaminated clothing and wash before reuse.
P332+P313 If skin irritation occurs: Get medical advice/attention.
P337+P313 If eye irritation persists: Get medical advice/attention.
Xn Harmful
Hazard-determining component of labeling: guanidinium hydrochloride
Risk phrases
R22 Harmful if swallowed.
R36/38 Irritating to eyes and skin.
Safety phrases
S23 Do not breathe gas/fumes/vapor/spray.
S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
S36/37 Wear suitable protective clothing and gloves.
S60 This material and its container must be disposed of as hazardous waste.
Warning
Hazard statements:
H302 Harmful if swallowed.
H315 Causes skin irritation.
H319 Causes serious eye irritation.
Precautionary statements:
P280 Wear protective gloves/protective clothing/eye protection/face protection.
P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy
to do. Continue rinsing.
P321 Specific treatment (see on this label).
P362 Take off contaminated clothing and wash before reuse.
P301+P312 IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell.
P501 Dispose of contents/container in accordance with local/regional/national/international regulations.
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its subsidiaries.
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