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Instruction Manual
PureLink™ 96 PlasmidPurification System
Catalog no. 12263-018
Version D 25 April 2005 54296
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iii
Table of Contents
Table of Contents.....................................................................................iii Kit Contents and Storage ..........................................................................1 Introduction...............................................................................................2
Methods .................................................................................. 4 Preparing 96-well Cultures .......................................................................4 Protocol A: Direct Load Method ..............................................................6 Protocol B: Harvested Cells Method ........................................................8
Appendix............................................................................... 10 Troubleshooting Guide ...........................................................................10 Applications ............................................................................................12 Media Selection Guide............................................................................14 Centrifugation Guide ..............................................................................15 Related Products .....................................................................................16 Purchaser Notification ............................................................................17 Technical Service....................................................................................18
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1
Kit Contents and Storage
Shipping and Storage
The PureLink™ 96 Plasmid Purification System components are shipped at room temperature. Upon receipt, store the kit at room temperature.
Kit Components
The PureLink™ 96 Plasmid Purification System contains the following components:
Component Composition Amount PureLink™ Growth Blocks — 4 PureLink™ Air Porous Tape — 4 PureLink™ 96 Filter Plates — 4 PureLink™ 96 Receiver Plates — 4 PureLink™ Foil Tape — 4 Cell Suspension Buffer 50 mM Tris-HCl (pH 8.0),
10 mM EDTA 120 ml
RNase A 20 mg/ml in 50 mM Tris-HCl (pH 8.0), 10 mM EDTA
2 × 1.5 ml
Lysis Buffer Proprietary 50 ml TE Buffer* 10 mM Tris-HCl (pH 8.0),
0.1 mM EDTA 30 ml
* The TE buffer provided contains very low EDTA, so it is compatible with sequencing and other enzymatic applications.
Additional Materials Needed
The following materials must be provided by the user: • Media (see page 4 for protocol requirements) • Sterile toothpicks • Appropriate antibiotics (see page 4 for concentrations) • Shaking incubator at 37°C • Isopropanol • 70% ethanol • Refrigerated centrifuge with a swinging bucket rotor with
plate carriers that have a plate height clearance of 6.2 cm. See page 15 for compatible models and rotors.
2
Introduction
Overview The PureLink™ 96 Plasmid Purification System offers a unique
approach to high throughput plasmid DNA isolation. A solid-phase lysis system replaces the numerous reagents and mixing steps associated with alkaline lysis. Cells are applied to a filter matrix within a 96-well plate, followed by addition of a lysis solution. During a short incubation, cells lyse within the matrix. Centrifugation extracts the plasmid DNA from the filter and precipitates the DNA in a receiver plate containing isopropanol. Genomic E. coli DNA and other cell components largely remain within the filter matrix.
Procedural Overview
This manual contains a cell growth procedure (see page 4) followed by two alternative purification procedures: • Protocol A, Direct Load Method, is for cell cultures grown
in rich media (e.g. Terrific Broth). This protocol is recommended for its higher yields and greater efficiency. See page 6.
• Protocol B, Harvested Cells Method, is for cells grown in Luria Bertani or similar media. See page 8.
System Features
The system has the following features and advantages: • Each protocol can be completed manually in 45–60
minutes. • Yields of 3–5 µg/well for Protocol A (Direct Load
Method) and 2–4 µg/well for Protocol B (Harvested Cells Method) are typical for high-copy-number plasmids.
• Plasmids up to 28 kb can be isolated and sequenced. • Filter plates can be processed 96 wells at a time or in
smaller increments. • Plasmid DNA isolated using this method is suitable for use
in automated fluorescent DNA sequencing, PCR, or restriction digest. (It is not recommended for use in transfection.)
• Cross-contamination of samples is eliminated by the procedural methods.
Continued on next page
3
Introduction, Continued
Instrument Compatibility
The PureLink™ 96 Plasmid Purification System is compatible with a variety of centrifuges, robots, and pipetting devices. See page 15 for more information on compatible centrifuges.
Critical Parameters
In the following protocols, the following parameters are critical: • Grow cultures with shaking at 300 rpm as directed by the
chosen protocol. • The filter plate/receiver plate assembly should be
centrifuged at 3,000 × g for the recommended time at 4°C. See page 15 for information on converting RPM to RCF (g), and modifications for lower g-force centrifuges.
• Excessive air-drying of samples from this system leads to nuclease-independent relaxation of supercoiled DNA. While evident on an agarose gel, this change in plasmid form does not affect performance.
4
Methods
Preparing 96-well Cultures
Cell Types While this system is compatible with various strains of E. coli,
DH10B cells are recommended for optimal performance and plasmid yield.
Required Media
The different protocols require different types of media: Protocol A, Direct Load Method — Rich media, such as Terrific Broth (Cat. no. 22711-022) or Super Broth. Protocol B, Harvested Cells Method — Luria Broth Base (Cat. no. 12795-027) or similar medium. See page 14 for media recipes and sources.
Antibiotic Concentration
The following antibiotic concentrations are recommended: • Ampicillin — 100 µg/ml • Chloramphenicol — 30 µg/ml • Gentamicin — 10 µg/ml • Kanamycin — 50 µg/ml • Streptomycin — 100 µg/ml • Tetracycline — 25 µg/ml
Continued on next page
5
Preparing 96-well Cultures, Continued
Preparing the Cell Cultures
Follow the steps below to prepare your growth block:
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1. Add an appropriate concentration of antibiotic to media (see previous page).
2. Place 1.3 ml of antibiotic-containing media into each well of a growth block. For Protocol A (Direct Load), the culture volume can be reduced to 0.5 ml if desired.
3. Using a sterile toothpick, touch a well-isolated colony (colonies should be no more than 2 weeks old). Drop the toothpick into a well of the growth block.
4. Repeat for all 96 wells of the growth block.
5. Remove toothpicks and discard into a biowaste container.
6. Cover growth block with air porous tape (supplied). Seal securely.
7. Incubate in a 37°C shaker at 300 RPM for 18–22 hours.
8. Check growth of a few random wells in the block by measuring A600 of a 1:10 dilution in TE. Be certain that cells are well suspended before removing aliquot. Use 100 µl of media plus 900 µl of TE as a blank. For optimal performance, A600 of a 1:10 dilution should be 0.4–0.8 for Protocol A (Direct Load) and 0.18–0.28 for Protocol B (Harvested Cells).
9. Proceed to Protocol A (next page) or Protocol B (page 8) as appropriate.
6
Protocol A: Direct Load Method
Preparing Lysis Buffer with RNase A
Immediately before using the protocol, add 0.6 ml RNase A per 12 ml lysis buffer in a separate conical tube. Invert 20 times to mix thoroughly. This quantity will be sufficient for one plate. Scale up as necessary for additional plates.
Protocol A
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1. Add 200 µl of isopropanol per well of a receiver plate.
2. Align a filter plate on top of the receiver plate. Keep the plates stacked through step 5. Do not place filter plate on absorbent surface for steps 4–6 or some sample may wick out.
3. If cells have settled in the growth block, mix cultures by pipetting up and down several times. Then transfer 200 µl of bacterial culture (OD600 4–8) to each well of the filter plate.
4. Add 100 µl lysis buffer containing RNase A to each well. Some of the liquid may not absorb into the filter. Do not mix. Incubate 10 min at room temperature.
5. Centrifuge the stacked plates at 3,000 × g for 15 min at 4°C. If a lower g-force must be used, see instructions on page 15 for adjustments to time.
6. Remove filter plate and discard.
Continued on next page
7
Protocol A: Direct Load Method, Continued
Protocol A, continued
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7. Drain liquid from receiver plate by inverting plate over a waste container and shaking briskly. Then invert plate on a stack of paper towels and tap to remove as much liquid as possible.
8. Add 500 µl 70% ethanol to each well of the receiver plate. Incubate at room temperature for 2 min.
9. Drain liquid from receiver plate as in step 7.
10. It is important to remove any remaining alcohol. Invert receiver plate onto a paper towel. Briefly centrifuge inverted plate by bringing the centrifuge to 35 × g, then immediately turning it off.
11. Air-dry plate for 5 min at room temperature.
12. Dissolve pellets by adding 25 µl TE buffer to each well. Allow plate to stand at room temperature or 4°C for at least 15 min. Cover the plate with foil tape and store at 4°C. Small quantities of insoluble material are sometimes present in the samples, but these do not affect performance.
Expect ~3-5 µg of DNA/well.
8
Protocol B: Harvested Cells Method
Preparing Lysis Buffer with RNase A
Immediately before using this protocol, add 0.6 ml RNase A per 12 ml lysis buffer. Invert 20 times to mix thoroughly. This quantity will be sufficient for one plate. Scale up as necessary for more plates.
Protocol B
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1. Harvest 1.0–1.3 ml of cells (OD600 1.8–2.8) by centrifuging the growth block at 1,500 × g for 10 minutes at 4–25°C.
2. Drain liquid from growth block by inverting plate on a stack of paper towels and tap to remove as much liquid as possible.
3. While cells are spinning, add 200 µl of isopropanol per well of a receiver plate.
4. Align filter plate on top of receiver plate. Keep the plates stacked through step 7. Do not place filter plate on absorbent surface for steps 5–7 or some sample may wick out.
5. Thoroughly resuspend cells in each well of the growth block with 200 µl cell suspension buffer. Complete resuspend-sion is critical, as clumps will reduce yield. Pipet cells up and down repeatedly or vortex on a plate vortexer to resuspend thoroughly.
6. Transfer cells to filter plate. Add 100 µl lysis buffer containing RNase A to each well of the filter plate. Some of the liquid may not absorb into the filter. Do not mix. Incubate 10 min at room temperature.
Continued on next page
9
Protocol B: Harvested Cells Method, Continued
Protocol B, continued
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7. Centrifuge the stacked plates at 3,000 × g for 15 min at 4°C. If a lower g-force must be used, see page 15 for adjustments to time.
8. Remove filter plate and discard. Drain receiver plate by inverting over a waste container and shaking briskly. Then invert plate on a stack of paper towels and tap to remove as much liquid as possible.
9. Add 500 µl 70% ethanol to each well of the receiver plate. Incubate at room temperature for 2 min.
10. Drain liquid from receiver plate as in step 8.
11. It is important to remove any remaining alcohol. Invert receiver plate onto a paper towel. Briefly centrifuge inverted plate by bringing the centrifuge to 35 × g, then immediately turning it off.
12. Air-dry plate for 5 min at room temperature.
13. Dissolve pellets by adding 25 µl TE buffer to each well. Allow plate to stand at room temperature or 4°C for at least 15 min. Cover the plate with foil tape and store at 4°C. Small quantities of insoluble material are sometimes present in the samples, but these do not affect performance.
Expect ~2.5 µg of DNA/well.
10
Appendix
Troubleshooting Guide Problem Possible Cause Suggested Solutions
Culture not dense enough
• For Protocol A: Direct Load Method use rich media such as Terrific Broth or Super Broth.
• For Protocols A and B: Incubate cultures 18–22 hours at 37°C.
• For Protocols A and B: Shake at 300 RPM during incubation to provide sufficient aeration.
High copy number plasmid behaves like a low copy number plasmid
• Include appropriate fresh antibiotic for plasmid vector at recommended antibiotic concentration (see page 4).
• Use DH10B cells to insure high plasmid DNA production.
• Check temperature of incubator to ensure it is at 37°C. Low incubation temperature can reduce plasmid copy number.
• After standard cleaning, rinse vessels used for preparing media with 95% ethanol followed by three rinses with deionized water to remove residual detergent. Traces of detergent in media can drastically reduce plasmid copy number, while cells will grow normally.
Low yield of plasmid DNA
Low copy number plasmid
• This product is not recommended for use with low copy number plasmids when the plasmid DNA is for use in automated fluorescent DNA sequencing. Low copy number plasmids can be used with this system when the samples are for use in PCR amplification.
Insufficient centrifugation • Centrifuge at 3,000 × g to elute and
precipitate DNA. Microplate carriers usually have a different radius from other buckets associated with a rotor. Be sure to calculate g-force using the radius of the rotor with the microplate carrier in place.
Loss of pellet • Centrifuge at 4°C for best adherence of pellet to well.
Continued on next page
11
Troubleshooting Guide, Continued Problem Possible Cause Suggested Solutions
Too many cells used • For Protocol A, Direct Load Method, cultures should be grown to OD600 of 4–8. If cultures are growing to higher density, reduce incubation time.
• For Protocol B, Harvested Cells Method, use only LB media. Cultures should be grown to OD600 of 1.8–2.8.
Temperature too high during centrifugation
• Ensure that centrifuge is running at 4°C or place centrifuge in a cold room.
Excessive insoluble material
Excessive centrifugation • Centrifuge at 3,000 × g to elute and precipitate DNA. Do not use higher g-force or longer times.
Weak fluorescence signal
DNA pellet insufficiently washed
• Perform a second 500 µl 70% ethanol wash before drying the DNA pellet. Be sure to perform the inverted plate centrifugation at the end.
Weak PCR amplification
DNA pellet insufficiently washed
• Perform a second 500 µl 70% ethanol wash before drying the DNA pellet. Be sure to perform the inverted plate centrifugation at the end.
Chromosomal DNA contamination
Too many cells used • For Protocol A, Direct Load Method, cultures should be grown to OD600 of 4–8. If cultures are growing to higher density, reduce incubation time in the future.
• For Protocol B, Harvested Cell Method, use only LB media. Cultures should be grown to OD600 of 1.8–2.8.
Excessive centrifugation • Centrifuge at 3,000 × g to elute and precipitate DNA. Do not use higher g-force or longer times.
RNA contamination
Insufficient RNase A • Add RNase A to lysis buffer immediately before use. Do not store reagent mix for more than one week.
12
Applications
Processing Fewer than 96 Samples
When fewer than 96 plasmid preps are required, you can use a portion of the filter plate. A partially used filter plate can be stored at room temperature and processed as many as 12 times without damaging the plate or the filters. However, each well can only be used once. To keep track of used wells and keep unused wells clean:
1. Cover the entire surface of the filter plate with foil tape.
2. Just prior to use, use a sharp blade to score the foil around the wells to be used and peel away the foil tape to expose the clean wells.
You will need a fresh receiver plate for each experiment. Additional receiver plates and foil tape can be purchased separately (see page 16).
DNA Quantitation
Plasmid DNA isolated using the PureLink™ 96 Plasmid Purification System is most easily quantitated using PicoGreen® (Molecular Probes) or Hoechst Dye 33258 in a 96-well format. A fluorescent plate reader is required for these techniques. Alternatively, you can quantitate the DNA by spectrophotometric measurement of the absorbance at 260 nm. However, the A260/A280 ratio cannot be used as an indicator of sample purity, because a small amount of a UV-absorbing component of the system co-purifies with the DNA. It does not interfere with automated fluorescent DNA sequencing, PCR amplification, or restriction digestion.
DNA Sequencing
Template quality and consistency are critical in automated fluorescent DNA sequencing. DNA templates prepared using the PureLink™ 96 Plasmid Purification System have been shown to be compatible with numerous sequencing chemistries, including BigDye™ terminator and ET primers. These templates are also compatible with a variety of automated DNA sequencers, including the ABI 3700, ABI 377, ABI 373, MegaBACE™, and LI-COR Global IR2. A second 70% ethanol wash can help increase fluorescence signal. In high throughput sequencing labs, templates prepared using PureLink™ 96 produced an average of 18% more bases with a Phred quality score of 20 or more (≥ 99% accurate) as compared to their previous plasmid purification method.
Continued on the next page
13
Applications, Continued
PCR High-throughput isolation of plasmid DNA for use as template
in PCR usually requires extra precautions and modified procedures to minimize cross-contamination. The PureLink™ 96 Plasmid Purification System procedure contains no mixing or vacuum filtration steps that can result in cross-contamination. Figure A below illustrates the absence of sample cross-contamination with the system.
In the figures, plasmid DNA was isolated from clones carrying two different-sized inserts. These clones were grown and purified from adjacent wells so that cross-contamination could be evaluated. Figure A shows results of PCR amplification with plasmid DNA isolated using PureLink™ 96 as the template. Figure B shows results from plasmid DNA isolated using a commercially available vacuum-based 96-well plasmid isolation system. The smaller clone (~ 400 bp insert) amplifies preferentially when the two clones are present in a mixture, allowing for detection of very small amounts of cross-contamination. The larger clone has an insert size of 2.4 kb.
Figure A PureLink™ 96
Figure B Vacuum-based
Controls
14
Media Selection Guide
Media Selection Guide
The table below lists recommended media and their recipes.
Terrific Broth (Cat. no. 22711-022)
11.8 g peptone 23.6 g yeast extract 9.4 g dipotassium hydrogen phosphate 2.2 g potassium dihydrogen phosphate 4 ml glycerol
Bring to 1 liter with deionized water and autoclave for 15 minutes at 121°C. Super Broth 32 g tryptone
20 g yeast extract 5 g sodium chloride Sufficient sodium hydroxide to adjust pH to 7.4
Bring to 995 ml with deionized water and autoclave for 15 minutes at 121°C, and then add 5 ml filter sterile 20% glucose. 2 × YT (acceptable, but less effective)
16 g peptone 10 g yeast extract 5 g sodium chloride
Bring to 1 liter with deionized water and autoclave for 15 minutes at 121°C. Luria Bertani, Miller’s Formulation (Cat. no. 12795-027)
10 g tryptone 5 g yeast extract 10 g sodium chloride (lower salt not recommended)
Bring to 1 liter with deionized water and autoclave for 15 minutes at 121°C.
15
Centrifugation Guide
Compatible Centrifuges and Rotors
Centrifuge and rotor combinations must be able to accom-modate a 6.2-cm microtiter plate stack. The following is a sampling of compatible equipment capable of reaching 3,000 × g.
Equivalent RPM Manufacturer Centrifuge Model Rotor Model
3000 × g 35 × g
IEC PR-7000M DoubleDeep 3,087 333 Jouan KR4.22 P60 3,160 341 Jouan CR4.12/CR4.22 M4 3,935 425 Jouan GR4.12/GR4.22 M4 3,935 425 Kendro (USA) Legend RT Swinging Bucket 4,104 443 Kendro (non-USA) Multifuge Swinging Bucket 4,104 443 Qiagen/Sigma Lab Centrifuges
4K15C
Plate Rotor
4,565
493
Converting RPM to RCF (g)
Use the following formula for converting RPM to RCF (g): RCF = 1.118 × Radius in mm × (RPM/1000)2
Where: RPM is the centrifuge speed in revolutions per minute, RCF is the relative centrifugal force (g), Radius is the distance (in mm) from the center of the centrifuge spindle to the bottom of the plate in the rotor when the microplate carrier is fully extended as during centrifugation.
Centrifugation at Lower g-Force
If an instrument for centrifuging the filter plate assembly is not capable of reaching 3,000 × g, longer centrifugation times can be used. See the table below for guidelines:
g-Force Centrifugation Time 3,000 15 min 2,500 20 min 2,000 25 min 1,500 30 min
16
Related Products
Related Products
The following products are also available from Invitrogen:
Product Amount Catalog No. PureLink™ 96 Components Available Separately
Growth Blocks pkg. of 50 12256-020 Air Porous Tape pkg. of 50 12262-010 Cell Suspension Buffer 500 ml 12056-016 Lysis Buffer 1 liter 12057-022 RNase A (20 mg/ml) 10 ml
25 ml 12091-021 12091-039
TE Buffer 100 ml 12090-015 Filter plates pkg. of 50 12192-035 Receiver Plates pkg. of 50 12193-025 Foil tape pkg. of 50 12261-012
Media Terrific Broth 500 g 22711-022 Luria Broth Base, powder (Miller's Luria Broth Base)
500 g
12795-027
Transformation and Testing ElectroMax™ DH10B™ Cells 5 × 0.1 ml 18290-015 ElectroMax™ DH10B™ T1 Phage Resistant Competent Cells
5 × 0.1 ml 12033-015
Max Efficiency® DH5α™ Competent Cells 5 × 0.2 ml 18258-012 Max Efficiency® DH5α™ T1 Phage Resistant Competent Cells
5 × 0.2 ml 12034-013
CloneChecker™ System 100 reactions 11666-013 Sequencing
Custom Primers—See our Web site (www.invitrogen.com) for information
17
Purchaser Notification
LLL No. 47: PureLink™ 96 and PureLink™ 384
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18
Technical Service
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Technical Service, Continued
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