JetQuick Plasmid Miniprep - Genomed

JetQuick Plasmid DNA Purication Kits
Rev. Date: 8 February 2011 Part no. 70-15012

MAN0003303
QUICK REFERENCE CARD
The JetQuick Plasmid DNA Purification Kit is designed to isolate up to 50 g of highquality plasmid DNA from E. coli cells in less than 20 minutes. To use the kit, lyse cells with alkaline/SDS and then apply the lysate to a silica membrane column that selectively binds plasmid DNA. Remove contaminants with Wash Buffers and elute the plasmid DNA in TE Buffer, Tris-HCl, orwater. Plasmid DNA purified with the JetQuick Plasmid DNA Purification Kit is suitable for all downstream applications.
Kit Components
Component 40-0050 Cell Resuspending Buffer (G1) RNase A Lysis Buffer (G2) Precipitation Buffer (G3) Optional Wash Buffer (GX) Wash Buffer (G4) TE Buffer Receiver Tubes JetQuick SpinColumn 14 mL 110 L 14 mL 20 mL 12 mL 12 mL 10 mL 50 each 50 each Catalog number 40-0250 69 mL 650 L 69 mL 96 mL 60 mL 60 mL 22 mL 250 each 250 each 40-0500 2 69 mL 2 650 L 2 69 mL 2 96 mL 2 60 mL 2 60 mL 2 22 mL 2 250 each 2 250 each 50 reactions 250 reactions 500 reactions
Safety Information
Buffers G3 and GX contain guanidine hydrochloride. Always wear a laboratory coat, disposable gloves, and eye protection when handling these buffers. Do not add bleach or acidic solutions directly to solutions or sample preparation waste containing guanidine hydrochloride as it forms reactive compounds and toxic gases when mixed with bleach or acids.
Before Starting

Add RNase A to the Cell Resuspending Buffer (G1) according to the label on the tube. Warm Lysis Buffer (G2) briefly at 37C to redissolve any particulate matter. Important: Add 96100% ethanol to Wash Buffer (GX) and Wash Buffer (G4) according to the labels on the tubes. Grow transformed E. coli in LB medium. Use 12 109 E. coli cells per preparation: 15 mL (high copy number plasmid) or 510 mL (low copy number plasmid) of an overnight culture. Note: Perform all centrifugations at >12,000 g in a conventional table-top microcentrifuge.
Intended Use For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Genomed
For technical support, email techservice@genomed-dna.com. For country-specic contact information, visit www.genomed-dna.com.
Purifying Plasmid DNA with a Centrifuge

1. Harvest. Grow 15 mL (high copy number plasmid) or 510 mL (low copy number plasmid) of transformed E. coli in LB medium overnight. Centrifuge the overnight LB-culture for23 minutes to pellet the cells. Remove all medium. 2. Resuspend. Add 250 L Cell Resuspending Buffer (G1) with RNase A to the pellet and resuspend the pellet until the suspension is homogenous. 3. Lyse. Add 250 L Lysis Buffer (G2). Mix gently by inverting the capped tube until the mixture is homogenous. Do not vortex. Incubate the mixture at room temperature for 5 minutes. 4. Neutralize. Add 350 L Neutralization Buffer (G3). Mix immediately by inverting the tube until the mixture is homogeneous. Do not vortex. Centrifugethe lysate for 10 minutes at room temperature. 5. Bind. Place a JetQuick SpinColumn into a provided 2-mL Receiver Tube. Pipet the supernatant from Step 4 onto the column. Centrifuge the column in the Receiver Tube for 1 minute. Discardtheflowthrough and place the column back in the Receiver Tube. 6. Optional Wash. Add 500 L Wash Buffer (GX) with ethanol to the column and centrifuge the column for 1 minute. Discardtheflowthrough and place the column back in the Receiver Tube. 7. Wash. Add 500 L Wash Buffer (G4) with ethanol to the column and centrifuge the column for 1 minute. Discardtheflowthrough. 8. Ethanol Removal. Place the column back in the Receiver Tube and centrifuge it again for 1 minute. Discard the Receiver Tube. 9. Elute. Place the column into a new 1.5-mL microcentrifuge tube. Add75 L 10 mM Tris-HCl, pH 8.0, TEBuffer, orwater to the column and centrifuge it for 2 minutes. 10. Store. The elution tube contains the purified plasmid DNA. Discard the column. Store the purified DNA at 4C for immediate use or aliquot the DNA and store it 20C for long-term storage.
Purifying Plasmid DNA using a Vacuum

1. Harvest. Grow 15 mL (high copy number plasmid) or 510 mL (low copy number plasmid) of transformed E. coli in LB medium overnight. Centrifuge the overnight LB-culture for23 minutes to pellet the cells. Remove all medium. 2. Resuspend. Add 250 L Cell Resuspending Buffer (G1) with RNase A and resuspend the pellet until the suspension is homogenous. 3. Lyse. Add 250 L Lysis Buffer (G2). Mix gently by inverting the capped tube until the mixture is homogenous. Do not vortex. Incubate the mixture at room temperature for 5 minutes. 4. Neutralize. Add 350 L Neutralization Buffer (G3). Mix immediately by inverting the tube until the mixture is homogeneous. Do not vortex. Centrifugethe lysate for 10 minutes at room temperature. 5. Bind. Attach a JetQuick SpinColumn onto a vacuum manifold. Pipet the supernatant from Step 4 into the column. Apply vacuum (200 to 650 mbar) until all of the supernatant has passed through the column and then turn off the vacuum source. 6. Optional Wash. Add 500 L Wash Buffer (GX) with ethanol to the column and incubate it for 1 minute. Apply vacuum (200to650mbar) until all of the supernatant has passed through the column and then turn off the vacuum source. 7. Wash. Add 500 L Wash Buffer (G4) with ethanol to the column. Apply vacuum (200 to 650 mbar) until all of the supernatant has passed through the column and then turn off the vacuum source.
8. Ethanol Removal. Place the column into a 2-mL Receiver Tube. Centrifuge the column in the tube for 1 minute. Discard the Receiver Tube. 9. Elute. Place the column into a new 1.5-mL microcentrifuge tube. Add75 L 10 mM Tris-HCl, pH 8.0, TEBuffer, orwater to the column and centrifuge it for 2 minutes. 10. Store. The elution tube contains the purified plasmid DNA. Discard the column. Store the purified DNA at 4C for immediate use or aliquot the DNA and store it 20C for long-term storage.
Troubleshooting
Problem Low plasmid DNA yield Solution Reconstitute Buffer G1 with RNase A and store Buffer G1 at 4C. Storeall other buffers at room temperature. Carefully remove all medium before resuspending cells. Increase the volume of starting culture. If the starting culture volume exceeds 5 mL, double the volumes of Buffers G1, G2, and G3. In this case, load the spin column with the neutralized supernatant, centrifuge it, discard the flow-through, and load the remaining neutralized supernatant onto the same spin column. Slow column flow Genomic DNA contamination Denatured plasmid DNA Ensure that the vacuum manifold is attached to a vacuum source and that unused luer extensions are closed. Gently invert tubes to mix after adding Buffers G2 and G3, respectively. Do not vortex during lysis or the initial precipitation as it can shear genomicDNA. Incubate the lysate, after the addition of Buffer G2, at room temperature for no longer than 5minutes.
Inhibition of Centrifuge the column to completely dry it or remove any downstream reactions remaining ethanol. Discard the flow-through. RNA contamination Make sure that RNase A is added to Buffer E1. Store Buffer E1 with RNase A at 4C for no longer than 6months. After 6 months, add fresh RNase A to Buffer E1.
Limited Use Label License: Research Use Only
The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchasers activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
Genomed
For technical support, email techservice@genomed-dna.com. For country-specic contact information, visit www.genomed-dna.com.

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JetQuick Plasmid DNA Purication Kits

Rev. Date: 8 February 2011 Part no. 70-15012

QUICK REFERENCE CARD

Purifying Plasmid DNA with a Centrifuge

Purifying Plasmid DNA using a Vacuum

Limited Use Label License: Research Use Only

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