CellRapeutics™ 96 Plasmid Kit (CART-018CL)

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Product Name
CellRapeutics™ 96 Plasmid Kit

Cat. No.
CART-018CL

Warnings
Buffer S2 contains NaOH, a caustic reagent. Buffer S3 and Buffer W1 contain chemical irritants.
When working with the buffers, always wear suitable protective clothing such as safety glasses,
laboratory coat and gloves. Be careful to avoid contact with eyes and skin. In the case of such contact,
wash immediately with water. If necessary, seek medical assistance.

General Description
The CellRapeuticsTM 96 Plasmid Kit represents a true high-throughput system for the purification of plasmid or cosmid DNA in a 96-well format. This product is designed to produce up to 20 µg of highly purified plasmid or cosmid DNA (per well) from up to 1.3 mL of bacterial cultures in 96-well format. Two plates rapidly and efficiently process the bacterial lysate, using either vacuum or centrifugation. The Lysate Filtration Plate efficiently removes bacterial cellular debris and transfers the clarified lysate directly into the Plasmid Plate for desalting. The plasmid is then eluted into a small volume of buffer or deionized water. The highly-purified plasmid or cosmid DNA is free from protein, genomic DNA and RNA and is suitable for any application, including: automated fluorescent sequencing on the ABI PRISM DNA Analyzers, in vitro transcription, restriction analysis and PCR. A vacuum manifold and vacuum source are required. High-throughput, 96-well plate format
Two-plate system ( lysate filtration and plasmid binding
Up to 20 µg of highly purified bacterial plasmid per well
Rapid vacuum and centrifuge protocols
No alcohol precipitation
Suitable for automated fluorescent sequencing
Automation-compatible plasticware

Components
Except for Buffer S1 (after addition of RNase A), all buffers are stable for a period of at least 12 months when stored under ambient conditions. Please avoid exposure to direct sunlight and extremes in temperature. To preserve RNase activity, the RNase A is suspended in a solution containing a high concentration of ammonium sulfate. On occasion, a precipitate may form. If this occurs, the precipitate is easily dissolved in Buffer S1 and the RNase activity is unaffected.

RNase A: 50 mg/ml. Store at room temperature.
Buffer S1: Resuspension buffer. Store at 4°C after addition of RNase A.
Buffer S2: Cell lysis buffer. Store at room temperature.
Buffer S3: Neutralization buffer. Store at room temperature.
Buffer W1: Wash buffer. Store at room temperature.
Buffer W2 concentrate: Desalting buffer. Add the amount of ethanol specified and store at room temperature. Either 100% or 95% denatured ethanol can be used.
10×Buffer W2 concentrate (only in 24×96 kit): Before use of the kit, add 15 ml of 10×Buffer W2, 135 ml of deionized water, 350 ml of ethanol to the empty 500 ml bottle supplied with the kit. Mix well and store at room temperature. Either 100% or 95% denatured ethanol can be used.
Eluent: 2.5mM Tris-HCl, pH 8.5. Store at room temperature.

Background Information
The CellRapeuticsTM 96 Plasmid Kit is suitable for the isolation of up to 20 μg of plasmid or cosmid DNA (per well) from up to 1.3 ml of bacterial culture. This method is based on a modified alkaline lysis of the bacteria in combination with the selective binding of the plasmid DNA to a special DNA plate. The purified plasmid or cosmid DNA is free from protein, genomic DNA and RNA and is suitable for a variety of applications, such as automated fluorescent sequencing, in vitro transcription, restriction analysis, PCR, eukaryotic cell transfection, etc. The CellRapeuticsTM 96 Plasmid Kit is ideal for use on automated sample preparation systems as a method for the high-throughput purification of plasmid intended for sequence analysis on platforms such as the ABI PRISM 3700 or 3730 DNA Analyzers. This kit is not recommended for the purification of large recombinant constructs, such as BAC and P1.

Preparation Note
Preparation before experiment 1) Supplement rich media with appropriate antibiotics.
2) Add one tube of RNase A into one bottle of Buffer S1, as described on the bottle label, and mix well. Use a volume of Buffer S1 to dissolve any precipitate and transfer it from the RNase A tube into the Buffer S1 bottle. Make a notation, including the date of RNase A addition to the Buffer S1 label.
3) Before the use of the kit, add the amount of ethanol specified on the bottle label into the Buffer W2 concentrate. Mix well. Either 100% or 95% (denatured) ethanol can be used.
4) Preparation of Buffer W2 (only in 24×96 kit). Add the following to the empty "Buffer W2 bottle" bottle provided in the kit:
15 ml of 10×Buffer W2 concentrate
135 ml of distilled water
350 ml of ethanol (100% or 95%)
5) Before each use, check Buffer S2 for precipitation. If precipitation occurs, warm in a 37°C water bath, accompanied by gentle swirling until the precipitate returns to solution. Equilibrate to room temperature before use.
6) Source some lint-free absorbent toweling.

Comments about the various protocol steps
Carefully read through the entire protocol, including the various "Notes" before beginning the procedure.
1. Bacterial Culture
This kit is designed for the isolation of high-copy plasmid or cosmid from 1.3 ml bacterial cultures, grown in rich media in a 96-well format. For the isolation of low-copy plasmids or cosmids containing pMB1 or ColE1 origins or replication, the culture should be supplemented with chloramphenicol according to standard protocols to increase the titer of plasmid. Please refer to the following table for details.

Bacterial cultures for plasmid isolation should always be grown from a single colony isolated from a freshly streaked plate. A single colony should be inoculated into rich media, such as LBG (Luria-Bertain broth+2% glycerol) or 2×YT containing the appropriate antibiotic. We recommend LBG for routine bacterial growth. TB is not recommended for bacterial growth and plasmid propagation. For bacterial growth, the kit includes 96-well 2.2 ml growblocks and BF-400 breathable film. The BF-400 breathable film is attached to the 96-well 2.2 ml growblock during growth to allow aeration of the bacterial cultures.
2. Collection of bacterial cells The bacteria are harvested by transferring the 2.2 ml growblock(s) to a benchtop centrifuge with a swinging bucket rotor and plate carriers and centrifuging at 4°C for 5 minutes at 1,500×g. At this g-force the bacterial cells will be efficiently pelleted and can also be easily resuspended in Buffer S1.
3. Cells lysis and neutralization After harvesting and resuspension, the bacterial cells are lysed by SDS under alkaline conditions. The lysis procedure should not extend beyond 5 minutes. Prolonged exposure to alkaline conditions may result in the formation of irreversibly denatured plasmid, which is refractory to many enzymatic reactions, such as restriction and sequencing. The alkaline lysate is neutralized by a high-salt buffer (S3) which also establishes the proper conditions for binding the plasmid to the DNA plate. During neutralization, the bacterial chromosomal DNA and cellular debris form a complex with the SDS, which is then precipitated out of solution by the high salt present in Buffer S3.
4. Removal of impurities by filtration and selective binding of DNA The insoluble debris complex, consisting of bacterial genomic DNA, bacterial cell wall debris and SDS/salt is removed by filtration through the Filtration plate. This plate contains specific porous materials which selectively remove this macroscopic debris complex while allowing the clarified filtrate containing the plasmid to pass through.
5. Washing, desalting and elution The membrane within the DNA plate is then washed consecutively with Buffer W1 and Buffer W2 to remove residual debris and to desalt the plasmid. After thoroughly removing all residual wash solution, the purified plasmid is eluted into a small volume of Tris solution and collected in a Vbottom sample plate. Large recombinant constructs, such as BAC and P1 often exhibit diminished elution efficiency and this kit is, therefore, not recommended for their purification.

Reagent and instrument requirements
• Heated water bath
• 8- or 12-channel pipette
• Disposable buffer trays
• Vortex
• Vacuum manifold or comparable model
• Vacuum source capable of -25-30 inches Hg
• Vacuum regulator
• Centrifuge with a swinging bucket rotor and plate carriers
• 100% or 95% (denatured) ethanol

Bacterial culture and collection
1. Fill each well of the 96-well 2.2 ml growblock with 1.3 ml of rich medium containing the appropriate antibiotic. Inoculate each well with a single bacterial colony and attach a piece of the BF-400 breathable film to the top of the 2.2 ml growblock. Incubate the cultures for 20-24 hours at 37°C with shaking at 250-300 rpm.
2. Centrifuge at 1,500×g for 5 minutes to pellet the bacterial cells. Discard the supernatant by inverting the 96-well growblock quickly. Invert the 2.2 ml growblock on a paper towel for 2 minutes to drain off the residual medium.

Cells lysis and neutralization
3. Add 0.3 ml of Buffer S1 containing RNase A to each well, and carefully seal the top of the 2.2 ml growblock with a piece of adhesive film. Resuspend the bacterial cells by vortexing. The bacterial suspension in each well should be completely homogenous with no visible aggregates.
Note: Make sure that RNase A has been added to Buffer S1.
Note: Incomplete bacterial resuspension will result in diminished lysis efficiency, plasmid yield and purity.
4. Remove the adhesive film and discard. Add 0.3 ml of Buffer S2 to each well and seal the top of the 2.2 ml growblock with a piece of silicon mat. Mix gently but thoroughly by inverting the 2.2 ml growblock 6-8×. This step should be completed within 5 minutes.
Note: It is important to mix gently when inverting the 2.2 ml growblock. Vigorous agitation will shear the genomic
DNA and result in contamination of the plasmid sample with genomic DNA fragments. If necessary, continue inverting the block until the solution becomes viscous and clear.
Note: After Buffer S2 is used, close the bottle immediately to avoid neutralization of the NaOH in Buffer S2 by
ambient CO2.
Note: Buffer S3 must be added within 5 minutes.
Note: The bacterial suspension will become viscous after lysis. To avoid cross-contamination between wells,
centrifuge briefly at 2,500×g before proceeding with the next step. Allow the centrifuge to reach 2,500×g, then stop.
5. Discard the silicone mat. Add 0.45 ml of Buffer S3 to each well and reseal the 2.2 ml growblock with a new silicone mat. Mix gently but thoroughly by inverting the block 6-8× or until a uniform precipitate is formed within each well. Incubate at room temperature for 5 minutes. Remove the silicone mat and discard.
Note: Do not shake or vigorously agitate the neutralized lysate.

Lysate filtration, plasmid binding and desalting
The Vacuum Manifold is required for this procedure.
6. Connect the vacuum manifold to the vacuum source. Remove the manifold top and place a 96-well V-bottom sample plate and an DNA plate into the manifold base. Replace the manifold top and place an Filtration plate onto the manifold top. Be sure that filtration plate drip directors are aligned with the wells of the DNA plate underneath (Please refer to Figure 1).
Note: It is advisable to connect a trap between the vacuum manifold and the vacuum source to avoid contamination and damage to the vacuum pump or source.

7. Using a pipetter, transfer as much lysate as is possible from Step 5 to the corresponding wells of the Filtration plate. Turn on the vacuum source and adjust it to -25-30 inches Hg. Continue to draw vacuum through the plates until no liquid remains in any of the wells of the Filtration plate. Turn off the vacuum source and release the vacuum from inside of the vacuum manifold using the vacuum regulator. Discard the Filtration plate. Remove the DNA plate from the manifold and temporarily set it aside on a piece of absorbent toweling or plastic wrap. Note: Be careful when handling the DNA plate. It will contain a substantial volume of clarified lysate. Note: Wash and save V-bottom sample plate aside for next use. 8. Place the waste collection tray into the manifold base. Replace the manifold top. Place the DNA plate onto the manifold. Turn on the vacuum and adjust to –25-30 inches Hg. Continue to draw the vacuum until no liquid remains in any of the DNA plate wells. Leave the vacuum on.
9. Optional step: Add 0.5 ml of Buffer W1 to each well of the DNA plate. Continue to draw the vacuum until no Buffer W1 is present in any of the wells.
Note: The use of Buffer W1 is only necessary to remove traces of endonuclease when endA+ bacterial strains have been used for plasmid propagation. These include the JM series and HB101. Host strains, such as XL-1 Blue and DH5α do not require a Buffer W1 wash.
10. Add 0.9 ml of Buffer W2 to each well. Apply vacuum until no Buffer W2 remains in any of the plate wells. Repeat the Buffer W2 wash.
Note: Make sure that ethanol has been added to the Buffer W2 concentrate.
11. Apply maximum vacuum (up to –30 inches Hg) for 10 minutes to remove residual ethanol.
Note: Residual ethanol from Buffer W2 may inhibit subsequent enzymatic reactions and must be removed completely.
12. Turn off the vacuum source. Remove the DNA plate from the vacuum manifold and vigorously tap the plate approximately 6-8× on several layers of absorbent toweling. Be careful not to damage the drip directors on the underside of the plate. Note: We recommend the use of "lint-free" absorbent toweling to avoid the release of tiny fibers which could
contaminate the plasmid and interfere with subsequent capillary electrophoresis.
Note: Alternatively, the DNA plate can be centrifuged to remove residual Buffer W2. Please follow the following
procedure: Turn off the vacuum. Place the DNA plate onto a 1.6 ml growblock. Transfer to a centrifuge with a swinging bucket rotor and plate carriers. Centrifuge at ≥ 3,000×g for 5 minutes.
Note: The kit contains a limited number of 1.6 ml growblocks. Save and use in subsequent preparations.

Elution
13. Remove the manifold top and place a 1.6 ml growblock into the manifold base. Place a Vbottom sample plate onto the 1.6 ml growblock. Reposition the manifold top and place the DNA plate on the manifold (Please refer to Figure 2).
Note: Be sure that the drip directors of the 96-well DNA plate are aligned directly over the wells of the V-bottom
sample plate.

14. Pipette 80-100 μl of Eluent or deionized water to the center of each well, and let it stand at room temperature for 1 minute. Turn the vacuum on and gradually increase the negative pressure to -15-30 inches Hg. Allow the vacuum to continue for 5 minutes.
Note: Pre-warming the Eluent to 65°C will generally improve elution efficiency.
15. Turn the vacuum off and use the vacuum regulator to gradually release the vacuum from the manifold. Disassemble the manifold and remove the V-bottom sample plate containing the purified plasmid samples.
Centrifuge-mediated elution (alternative)
Place the DNA plate on a V-bottom sample plate. Pipette 80 μl of Eluent or deionized water to the center of each well, and let it stand at room temperature for 1 minute. Centrifuge at ≥3,000×g for 5 minutes.

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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