LaboPassTMMini

Protocol Book

LaboPass TM Plasmid Mini
Introduction
LaboPass TM Plasmid Mini kit provides easy and rapid method for the smallscale preparation of plasmid DNA from bacterial cells. This kit can be used toisolate and purify any plasmid, but works most efficiently when the plasmid isless then 20,000 bp in size. All process to prepare pure plasmid DNA takes
only about 30 min and simultaneous processing of multiple samples can beeasily performed. Up to 30 µg of pure plasmid can be purified using LaboPassTMPlasmid Mini kit and this pure plasmid DNA is ready for PCR, cloning,fluorescent sequencing, synthesis of labeled hybridization probes, celltransfection, electroporation, and enzymatic restriction without furthermanipulation.

Principle of method
LaboPass TM Plasmid Mini kit utilizes glass microfiber membrane based on themodified alkaline lysis method. Alkaline lysis releases plasmid DNA frombacterial cells and RNase A removes any RNA in the lysate. In the presence ofhigh salt, plasmid DNA binds selectively to glass microfiber membrane in theLaboPass TM spin column. Bound plasmid DNA is purified in a series ofwashing steps to eliminate contamination of other bacterial components.
Finally elution by low salt buffer or water releases plasmid DNA from the glass
microfiber membrane. This simple method eliminates the need for organic
solvent extractions and alcohol precipitation, allowing for rapid purification ofmany samples simultaneously.

Storage and Stability
LaboPass TM Plasmid Mini kit is shipped at room temperature. All components
except RNase A solution are stable at room temperature for 12 months fromthe date of manufacture that is printed on the product package. RNase A stock
solution is stable at room temperature, but for longer conservation of enzyme
activity, storage at ４℃ is recommended. After addition of RNase A solution,
Buffer S1 is stable for 6 months when stored at 4℃. In cold ambient conditions,
Buffer S2, S3 and AW may exhibit salt precipitation and this causes reductionof DNA yields. If so, heat the bottles to dissolve completely at 60℃.

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Chemical Hazard
Buffer S3 and AW contain irritant which is harmful when in contact with skin, or
when inhaled or swallowed. Care should be taken during handling. Always
wear gloves and eye protector, and follow standard safety precautions.

Quality Control
All components in LaboPass TM Plasmid Mini kit are manufactured in particlecontrolled area (class 1000 clean room), and its degree of cleanness ismonitored periodically.

Kit Contents
Cat. No.
No. of preparation
Spin column
Collection tube
Buffer S1(1)Buffer S2
Buffer S3
Buffer AW (conc.)
Buffer PW (conc.)
Buffer EB
Rnase A solution (100mg/ml)

CMP0111
5050
50
15ml
15ml
20ml
17.5ml
15ml
15ml
15µl

CMP011220020020055ml
55ml
80ml
70ml
57ml
15ml
55µl

CMP0115
1000
1000
1000
55mlx555mlx580mlx570mlx257mlx515mlx555µl x5(1) Buffer S1 should be stored in 4℃ after additional of RNase A solution.
* All Solution (Buffer) is available to purchase separately.

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LaboPass TM Plasmid Mini kit Protocols
Before experiment- Before first use, add appropriate volume of absolute ethanol to Buffer AW and
PW as printed on the product label.
- All centrifugation steps should be performed at full speed (>10,000 xg or 10,000 14,000 rpm) in a microcentrifuge at room temperature.
- Mix Buffer S2 and S3 thoroughly by shaking before use.
- Buffer S2 or S3 may precipitate at cool ambient conditions. If so, dissolve it in 60 ℃
water bath.

1. Pellet the bacterial culture by centrifugation for 5 min at 10,000xg in a tabletop centrifuge. Discard the supernatant as much as
possible.
Use the appropriate volume of bacterial cultures; 1-5 ml for high copy number
plasmid, 1-10 ml for low copy number plasmid. Bacterial culture should begrown for 12 to 16 hours in liquid media (eg. LB) containing a selective
antibiotic to a density of A600 =2.0. Higher culture volumes can cause reductionof lysis efficiency and overload of a spin column, resulting in unsatisfactory
yields.

2. Resuspend pelleted bacterial cells thoroughly in 250μl of Buffer
S1. Transfer the suspension to a new 1.5 ml tube.
Add RNase A solution before the first use of the Buffer S1. It is essential tothoroughly resuspend the cells.

3. Add 250μl of Buffer S2 and mix by inverting the tube 4 times (donot vortex).
Incubate until the cell suspension becomes clear and viscous; approximately
for 1-5 min. Do not incubate for more than 5 min. If precipitated material has
formed in buffer S2, heat to dissolve at 37℃

4. Add 350μl of Buffer S3 and immediately mix by inverting the tube
4-6 times (do not vortex)
For better precipitation, mix the solution gently but completely and immediately
after addition of buffer S3.

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5. Centrifuge for 10 min.
6. Transfer carefully the supernatant to a spin column andcentrifuge for 1 min. Remove the spin column, discard theflowthrough, and re-insert the spin column to the collection tube.
7. When using endA+ strains, continue with step 7A, 7B. If not,continue with step 7C.
The wild type E.coli strains produce endonuclease I, which is encoded in endA
gene and degrades double-stranded DNA. The E.coli genotype endA1 refers
to a mutation in the wildtype endA gene, which produces an inactive form of
the nucelase. E.coli strains with this mutation are referred to as endA-. The
absence of endA1 (or endA) in an E.coli genotype denotes the presence of thewildtype gene, which expressed an active endonuclease I. The wildtype isindicated as endA+. Buffer AW of step 7A removes the trace of endonuclease
when using endA+ strains. If you cannot ensure the endA genotype of E.coli,
follow the procedure of endA+. (step 7A and 7B)

The genotype of various E.coli strains
EndA+ strains; BL21(DE3), CJ236, HB101, JM83, JM101,JM110, LE392, MC1061, NM series, P2392, PR series, RR1,TB1, TG1, BMH71-18, ES1301, wildtype, and etcEndA- strains; BJ5183, DH1, DH20, DH21, DH5
JM105, JM106, JM107, JM108, JM109, MM294, SK1590, SRB,XL1-Blue, XLO, and etc.
endA+ strains
7A. Apply the 500μl of Buffer AW and centrifuge for 30 sec.
Remove the spin column, discard the flowthrough, and re-insert thespin column to the collection tube.
7B. Apply the 750μl of Buffer PW and centrifuge for 1 min. Remove
the spin column, discard the flowthrough, and re-insert the spincolumn to the collection tube. Go to step 8.
endA- strain
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7C. Apply the 750μl of Buffer PW and centrifuge for 1 min.
Remove the spin column, discard the flowthrough, and re-insert thespin column to the collection tube.
8. Discard the flowthrough, and centrifuge for an additional 1 min toremove residual wash buffer. Transfer the spin column to a new 1.5
ml tube.
If the spin column has Buffer PW associated with it, centrifuge again for
additional 1 min at full speed before transferring to the new 1.5 ml tube.
Residual ethanol from Buffer PW may inhibit subsequent enzymatic reaction.

9. Add 50μl of buffer EB or deionized distilled water, let stand for 1min, and centrifuge for 1 min.
Ensure that the buffer EB or distilled water is dispensed directly onto thecenter of spin column membrane for optimal elution of DNA. For high copy
number plasmids, elution volume can be increased to 200 µl maximum. It will
increase the total yields of plasmid but decrease the concentration of eluate.
For long-term storage, eluting in buffer EB(10mM Tris-Cl, pH 8.5) and storingat –20℃ is recommended. When using water for elution, check the pH ofwater (>pH7.0) before elution.

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Troubleshooting guide
Low or no yield of plasmid DNA-Too many cells in sample
Cultures should be grown to A600=2~4 in proper media with antibiotics. Reducethe culture volume of sample. (For low-copy number plasmid, 10ml, for highcopy plasmid, 5ml maximum)- Low-copy-number plasmid used
Low-copy-number plasmids may yield as little as 0.5 µg of DNA from a 5 ml
overnight culture. Increase culture volume to 10 ml or use high copy-number
plasmid, if possible.
- Poor resuspension of bacterial pellets in Buffer S1Bacterial cell pellets ought to be resuspended throughly in Buffer S1.
- Buffer S2 precipitated
Dissolve Buffer S2 by warming to 37℃.
- Ethanol was not added to Buffer AW and PW
Buffer AW and PW should be diluted with appropriate volume (printed on alabel) of absolute ethanol.
- Insufficient digestion with RNase A
Excess RNA can interfere the binding of plasmid DNA with glass-fiber
membrane. If Buffer S1 containing RNase A is more than 6 months old, add
additional RNase A solution. (Working concentration = 100µg /ml)
Low or no yield of plasmid DNA- Inadequate elution buffer
DNA is eluted only in low salt condition. Buffer EB (10mM Tris-Cl, pH8.5) has
the optimal elution efficiency, but other elution buffer can be engaged as user'sneed. Elution efficiency is dependent on pH and the maximum efficiency isachieved between pH 7.0 and pH 8.5. When using water for elution, makesure the pH value.
Chromosomal DNA contamination- Mishandling of the lysate after addition of Buffer S2Vigorous vortexing after addition of Buffer S2 can cause shearing ofchromosomal DNA followed by chromosomal DNA contamination. Handle
gently the lysate after addition of Buffer S2. Simple inverting and rotating tube
to cover walls with viscous lysate is sufficient for mixing.
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- Too long lysis time
Too long lysis time after addition of Buffer S2 can cause chromosomal DNA
contamination. Lysis time should not be over 5 min. And immediate mixing
after addition of Buffer S3 is strongly recommended.
RNA contamination- RNase A omitted or old
RNase A solution should be added to Buffer S1 before first use. If Buffer S1containing RNase A is more than 6 months old, add additional RNase solution.
(work conc.=100 µg /ml)
- Too many cells in sample
Reduce the sample volume. Too many cells may not be subjected properly toRNase A digestion.
High salt concentration in eluate- Improper wash step
Ensure the wash step in protocols. Alternatively, incubate for 5 min at room
temperature after applying Buffer PW in step 7B(endA+) or 7C(endA-).
Plasmid DNA degradation- Nuclease contamination
For endA+ strains, stick to the protocols for endA+ strains. Buffer AW ought tobe used in wash step.
DNA floats out of well while loading of agarose gel- Ethanol is not completely removed during wash steps.
Ensure that step 8 in protocols is performed well. Spin column membrane
should be completely dried via additional centrifugation or air-drying. Refer theannotation of step 8 in protocols.

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Contact us ;製造元

COSMO GENETECH CO.,Ltd.

Internet : www.cosmo4.com
E-mail : LaboPass@cosmo4.com
Tel : 82-2-922-7307Fax : 82-2-922-7307

販売元
北海道システム・サイエンス株式会社

HokkaidoSystem Science.,Ltd.
北海道システム・サイエンス株式会社〒001-0932
札幌市北区新川西2条1丁目2-1
TEL : 011-768-5901
FAX : 011-768-5951
E-mail : bio@hssnet.co.jp
URL : http://www.hssnet.co.jp/

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