Dec 14, 2016
SpinSmart Plasmid Purification: Low-copy plasmid DNA, P1 constructs, or cosmids
- Denville Scientific
- Denville Scientific, Inc.
External link: https://www.denvillescientific.com/products/spinsmart%E2%84%A2-plasmid-miniprep-dna-purification-kit-with-reagents--50-preps
Protocol Citation: Denville Scientific 2016. SpinSmart Plasmid Purification: Low-copy plasmid DNA, P1 constructs, or cosmids. protocols.io https://dx.doi.org/10.17504/protocols.io.gribv4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: December 14, 2016
Last Modified: November 09, 2017
Protocol Integer ID: 4618
Abstract
SpinSmart Plasmid Miniprep kits are designed to rapidly purify plasmid DNA from bacterial cultures.
Guidelines
Spin Smart Plasmid Miniprep Kit Components
Equipment to be supplied by user
96-100% ethanol
1.5 ml microcentrifuge tubes
Manual piEquipment to be supplied by userpettors and disposable pipette tips
Centrifuge for microcentrifuge tubes
Personal protection equipment (lab coat, gloves, goggles)
Product description
SpinSmart Plasmid Miniprep kits are designed to rapidly purify plasmid DNA from bacterial cultures. Bacteria are grown overnight in culture media, then harvested by centrifugation. Pelleted bacterial cells are resuspended in PB1 buffer, then alkaline lysis buffer PB2 releases plasmid DNA from the cells. The reaction is neutralized by PB3 buffer, which also creates appropriate conditions for binding plasmid DNA to the SpinSmart Plasmid Miniprep silica membrane. A centrifugation step pellets precipitated protein, genomic DNA, and cell debris. The supernatant is loaded onto a SpinSmart Plasmid Miniprep binding column.
Salts, metabolites, and soluble cellular debris are removed by wash steps with ethanolic PB5 Wash Buffer. After the wash steps, plasmid DNA is eluted from the column with PB6 Elution Buffer (5 mM Tris/HCl, pH 8.5). If host strains with high levels of nucleases are used, we recommend an additional washing step with pre-warmed PB4 Wash Buffer. The additional wash with PB4 will also increase the reading length of automated fluorescent DNA sequencing reactions.
SpinSmart Plasmid Columns have a DNA binding capacity of 60 µg.
SpinSmart purified plasmid DNA is suitable for applications like automated fluorescent DNA sequencing, PCR, or other enzymatic reactions.
Growth of bacterial cultures
Plasmid DNA quality and yield is highly dependent on the type of culture media and antibiotics used, the bacterial host strain, the plasmid type, size, and copy number.
LB (Luria Bertani) medium is recommended for standard, high-copy plasmids. The cell culture should be incubated at 37°C with constant shaking (200-250 rpm) preferably 12-16 h overnight. The culture should be grown to an OD600 of 3-6. Rich media, such as 2xYT (Yeast/Tryptone), TB (Terrific Broth) or CircleGrow, can also be used. Bacteria grow faster in rich media, so they reach the stationary phase much earlier than in LB medium (≤ 12 h), and they also achieve higher cell masses. However, this does not necessarily yield more plasmid DNA. Overgrowing a culture could lead to a high percentage of dead or starving cells, which can result in plasmid DNA that is partially degraded or contaminated with chromosomal DNA. Culture medium and incubation time must be optimized for each host strain/plasmid construct combination individually.
Cell cultures must be grown under antibiotic selection to ensure plasmid propagation.
∗ Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor, Cold Spring, New York 1982.
SpinSmart Plasmid Minipreps recommend to use 5 ml of a well grown bacterial culture, OD600 = 3.
Culture volumes can be increased if the cell culture grows very poorly or has to be decreased. Please note the OD600 values and corresponding culture values below:
| Recommended Culture Volumes | |||||||
| OD600 | 1 | 2 | 3 | 4 | 5 | 6 | |
| Culture Volume | 15 ml | 8 ml | 5 ml | 4 ml | 3 ml | 2 ml |
Elution procedures
High yield: important for larger constructs: Heat elution buffer to 70°C, add 50-100 µl to the SpinSmart Plasmid miniprep column. Incubate at 70°C for 2 min.
High yield: Perform two elution steps with the volume indicated in the individual protocol. 90-100% of bound plasmid DNA can be eluted.
High concentration: Perform one elution step with 60% of the volume indicated in the individual protocol.
High yield and high concentration: Apply half of the volume of elution buffer as indicated in the individual protocol, incubate for 3 min and centrifuge. Apply a second aliquot of elution buffer, incubate and centrifuge again.
SpinSmart Plasmid Miniprep protocols recommend PB6 Elution Buffer (5 mM Tris, pH 8.5) for elution. We do not recommend buffers containing EDTA, especially if the plasmid DNA is intended for sequencing reactions. Water may be used for elution, however the pH should be adjusted to pH 7.0-8.5 since deionized water typically has a low pH.
Storage and preparation of solutions
Buffers PB3 and PB4 contain guanidine hydrochloride! Wear gloves and goggles when using this kit!
All kit components can be stored at room temperature (20-25°C) and are stable for up to one year.
Keep buffer bottles tightly closed, especially if buffers are warmed at any time.
Sodium dodecyl sulfate (SDS) in PB2 Lysis Buffer may precipitate if stored at temperatures below 20°C. If a precipitate is observed, incubate the bottle at 30–40°C for several minutes and mix well.
Prior to starting the SpinSmart Plasmid Miniprep prepare the following:
Before the first use of the kit, add 1 ml of PB1 Resuspension Buffer to the RNase A vial and vortex briefly. Transfer the mixture into the PB1 bottle and mix thoroughly.
Store PB1 Resuspension Buffer (containing RNase A) at 4°C for up to 6 months.
Add the indicated volume of 96-100% ethanol to PB5 Wash Buffer.
| Catalog Number | 50 preps Cat. No. CM-410-50 | 250 preps Cat. No. CM-410-250 | |
| Buffer PB5 (Concentrate) | 2 x 6 ml add 24 ml 96% - 100% EtOH | 2 x 20 ml add 80 ml 96% - 100% EtOH |
Safety Information
Wear gloves and goggles and follow the safety instructions given in this section.
Risk Phrases
R 10 Flammable
R 22 Harmful if swallowed
R 36/38 Irritating to eyes and skin
R 42/43 May cause sensitization by inhalation and skin contact
Safety Phrases
S 7 Keep container tightly closed
S 16 Keep away from sources of ignition - No Smoking!
S 22 Do not breathe dust
S 24 Avoid contact with the skin
S 25 Avoid contact with the eyes
S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
S 37/39 Wear suitable protective clothing and gloves
S 45 In case of accident or if you feel unwell, seek medical advice immediately
(show the label where possible)
∗ Label not necessary, if quantity below 25 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)
∗∗ Label not necessary, if quantity below 125 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)
Troubleshooting
Please see the product manual for troubleshooting information.
Materials
MATERIALS
SpinSmart™ Plasmid Miniprep DNA Purification Kit with Reagents, 50 prepsDenville Scientific Inc.Catalog #CM-410-50
Safety warnings
Buffers PB3 and PB4 contain guanidine hydrochloride! Wear gloves and goggles when using this kit!
Safety Information
Wear gloves and goggles and follow the safety instructions given in this section.
Risk Phrases
R 10 Flammable
R 22 Harmful if swallowed
R 36/38 Irritating to eyes and skin
R 42/43 May cause sensitization by inhalation and skin contact
Safety Phrases
S 7 Keep container tightly closed
S 16 Keep away from sources of ignition - No Smoking!
S 22 Do not breathe dust
S 24 Avoid contact with the skin
S 25 Avoid contact with the eyes
S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
S 37/39 Wear suitable protective clothing and gloves
S 45 In case of accident or if you feel unwell, seek medical advice immediately
(show the label where possible)
∗ Label not necessary, if quantity below 25 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)
∗∗ Label not necessary, if quantity below 125 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)
Before start
Prior to starting the SpinSmart Plasmid Miniprep prepare the following:
Before the first use of the kit, add 1 ml of PB1 Resuspension Buffer to the RNase A vial and vortex briefly. Transfer the mixture into the PB1 bottle and mix thoroughly.
Store PB1 Resuspension Buffer (containing RNase A) at 4°C for up to 6 months.
Add the indicated volume of 96-100% ethanol to PB5 Wash Buffer.
| Catalog Number | 50 preps Cat. No. CM-410-50 | 250 preps Cat. No. CM-410-250 | |
| Buffer PB5 (Concentrate) | 2 x 6 ml add 24 ml 96% - 100% EtOH | 2 x 20 ml add 80 ml 96% - 100% EtOH |
Cultivate and harvest bacterial cells
Cultivate and harvest bacterial cells
Start with 5-10 ml E. coli LB culture*, pellet cells in a microcentrifuge for 30 sec at 11,000 x g
00:00:30
Note
Culture should be grown to OD600 3-6. SpinSmart Plasmid Minipreps recommend to use 5 ml of a well grown bacterial culture, OD600 = 3.
Culture volumes can be increased if the cell culture grows very poorly or has to be decreased. Please note the OD600 values and corresponding culture values below:
| Recommended Culture Volumes | |||||||
| OD600 | 1 | 2 | 3 | 4 | 5 | 6 | |
| Culture Volume | 15 ml | 8 ml | 5 ml | 4 ml | 3 ml | 2 ml |
Discard the supernatant and remove as much of the liquid as possible.
Cell lysis
Cell lysis
Add 500 µl PB1 Resuspension Buffer.
Vortex or pipet up and down to resuspend the cell pellet completely. No cell clumps should be visible.
Note
Important: Buffer PB2 should not have SDS precipitate visible prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30-40°C until precipitate is dissolved completely. Cool buffer down to room temperature (20-25°C).
Add 500 µl PB2 Lysis Buffer.
Invert the tube 6-8 times to mix completely. Do not vortex!
Note
Do not vortex!
Incubate at room temperature for up to 5 min or until lysate appears clear.
00:05:00
Add 600 µl PB3 Neutralization Buffer
Invert the tube 6-8 times to mix completely. Do not vortex!
Note
Do not vortex!
Lysate Clarification
Lysate Clarification
Centrifuge for 10 min at 11,000 x g at room temperature.
Note
5 - 10 min ≥ 11,000 x g
If precipitate is not completely clear from the lysate, centrifuge again for 10 min at 11,000 x g at room temperature.
Bind DNA
Bind DNA
Place a SpinSmart Plasmid Binding Column in a Collection Tube (2 ml) and load a maximum of 750 µl of the supernatant (from the "Lysate Clarification" section above) onto the column.
Centrifuge for 1 min at 11,000 x g.
00:01:00
Discard flow-through and place the SpinSmart Plasmid Binding Column into the Collection Tube (2 ml).
Note
Repeat steps 12-14 of this section to load any remaining lysate.
Wash silica membrane
Wash silica membrane
Add 500 µl PB4 Wash Buffer pre-warmed to 50°C.
Centrifuge for 1 min at 11,000 x g before proceeding with Buffer PB5.
00:01:00
Discard flow-through and place the SpinSmart Plasmid Column back into the empty Collection Tube (2 ml).
Add 600 µl PB5 Wash Buffer (make sure EtOH has been added).
Centrifuge for 1 min at 11,000 x g.
00:01:00
Discard flow-through and place the SpinSmart Plasmid Column back into the empty Collection Tube (2 ml).
Dry silica membrane
Dry silica membrane
Centrifuge for 2 min at 11,000 x g and discard the Collection Tube (2 ml).
00:02:00
Elute highly pure DNA
Elute highly pure DNA
Place the SpinSmart Plasmid Binding Column in a 1.5 ml microcentrifuge tube (not provided) and add 50 µl PB6 Elution Buffer pre-warmed to 70°C..
Note
SpinSmart Plasmid Miniprep protocols recommend PB6 Elution Buffer (5 mM Tris, pH 8.5) for elution. We do not recommend buffers containing EDTA, especially if the plasmid DNA is intended for sequencing reactions. Water may be used for elution, however the pH should be adjusted to pH 7.0-8.5 since deionized water typically has a low pH.
Incubate for 2 min at 70°C.
00:02:00
Centrifuge for 1 min at 11,000 x g.
00:01:00