for purification of DNA from PCR reactions Cat. No. 1 73 668 (50 purifications) Cat. No. 1 73 676 (50 purifications) Principle In the presence of chaotropic salt, product DNA binds selectively to glass fiber fleece in a special centrifuge tube. The DNA remains bound while a series of rapid wash-and-spin steps remove contaminating small molecules (including small nucleic acids). Finally, low salt elution removes the DNA from the glass fiber fleece. The process does not require DNA precipitation, organic solvent extractions, or extensive handling. Starting material Application Time required Results Key advantages Samples (100 µl) may be: DNA products (> 100 bp 50 kb) from PCR Enzymatically labeled, modified, or digested DNA (for example, products from restriction digests, alkaline phosphatase treatments, kinase reactions, or enzymatic labeling reactions) RNA from in vitro transcription reactions* Note: The kit may also be used to prepare DNA from a 100 mg agarose gel slice. Preparation of concentrated, purified DNA, which may be used directly for sequencing, cloning and other routine applications Total time: approx. 10 min Hands-on time: < 10 min Yield: Variable, depending on sample volume and DNA size (See the table under Part IV of How to use the kit in this article) Purity: Purified DNA is free of short DNA (< 100 bp), small molecules (for example, mineral oil, primers, salts, unincorporated nucleotides) and proteins (for example, thermostable enzymes) Saves time, because the kit makes PCR products ready for downstream procedures in less than 10 min Minimizes DNA loss, because the kit removes contaminants without precipitation or other handling steps that lead to DNA loss or degradation Increases lab safety, because the kit does not use hazardous organic solvents Accommodates a wide variety of samples, because the kit can purify DNA from most enzymatic reaction mixtures, DNA from agarose gels, even RNA from in vitro transcription reactions Improves performance of downstream applications, because the kit removes DNA fragments smaller than 100 bp from the preparation * According to a customer, the kit may be used to remove unincorporated label from transcribed RNA 0
How to use the kit I. Flow diagram The steps in this kit are similar to those of other High Pure kits. For a general flow diagram showing how the High Pure kits work, see page 11 in this manual. II. Kit contents Binding Buffer with guanidine thiocyanate (30 ml or 150 ml) Wash Buffer (10 ml or 50 ml) Note: Add absolute ethanol (40 ml or 00 ml) to Wash Buffer before use Elution Buffer (TE buffer, ph 8.5) (30 ml) High Pure Filter Tubes (50 or 50) Collection Tubes, ml (50 or 50) Note: Both sizes of the kit contain the same components; only the amount (values in parentheses) of the components in the kit changes. III. Additional materials needed Absolute ethanol Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force Microcentrifuge tubes, 1.5 ml, sterile Agarose TAE buffer (40 mm Tris-acetate, 1 mm EDTA), ph 8.0 Electrophoresis equipment Sterile scalpel Isopropanol IV. Expected DNA recovery from various amounts and sizes of DNA DNA applied ( g) Recovery (%) Fragment length (bp) Recovery (%) Elution volume ( l) Recovery (%) 5 77 < 100 < 5 50 68 10 79 375 > 95 100 79 5 80 700 > 95 150 80 50 56 3000 > 95 00 80 Note: The amount of DNA recovered also varied with the volume of Elution Buffer applied in the recovery step: 1
V. Protocols for preparing DNA Va. Purifying amplification products from 100 l PCR product mix Note: To process a larger sample (> 100 l), either increase proportionally the amount of Binding Buffer (Step 1) or divide the large sample into several aliquots and process the aliquots as separate samples. 1 After the PCR amplification is complete, adjust the volume in one PCR tube (reaction components + DNA product) to 100 µl, then: 3 4 5 6 Add 500 µl Binding Buffer to the contents (100 µl) of the PCR tube. Note: You do not need to remove mineral oil or wax from the PCR solution before adding the Binding Buffer. Mix the sample (Binding Buffer + starting PCR solution) well. After mixing the sample: Insert one High Pure Filter Tube into one Collection Tube. Pipette entire sample into upper buffer Insert the entire High Pure Tube assembly into a standard tabletop microcentrifuge, then centrifuge the tube assembly for 30 s at full speed (approx. 13,000 x g). After the centrifugation: Remove the Filter Tube from the Collection Tube. Discard the flowthrough liquid in the Collection Tube. Reinsert the Filter Tube in the same Collection Tube. To wash the sample: Add 500 µl Wash Buffer to the upper Repeat the centrifugation (as in Step 3). After the first wash: Discard the flowthrough and recombine the tubes (as in Step 4). Add 00 µl Wash Buffer to the upper Repeat the centrifugation (as in Step 3). 7 8 9 After the second wash: Remove the Filter Tube from the Collection Tube. Discard the Collection Tube (with the flowthrough liquid). Insert the Filter Tube into a clean, sterile 1.5 ml microcentrifuge tube. To elute the DNA from the Filter Tube: Add 50 100 µl Elution Buffer or redistilled water (ph 8.0 8.5) to the upper Note: For optimal recovery of DNA, use 100 l buffer or water. Centrifuge the tube assembly for 30 s at full speed. The microcentrifuge tube now contains the eluted DNA. You may: EITHER use the eluted DNA directly in such applications as cloning or sequencing OR store the eluted DNA at 4 C for later analysis Vb. Purifying DNA from a 100 mg slice of agarose gel 1 Purify the DNA of interest electrophoretically as follows: Prepare an agarose gel with Boehringer Mannheim agarose (MP, LE, MS, or LM-MP) and load the DNA of interest on the gel. Use 1 x TAE (40 mm Tris-acetate, 1 mm EDTA, ph 8.0) or 1 x TBE (45 mm Tris-borate, 1 mm EDTA, ph 8.0) as running buffer. Electrophorese until the DNA of interest is well separated from contaminants. After gel electrophoresis, stain the gel with ethidium bromide. 3 Cut the DNA band from the gel with a sharp scalpel or razor blade. Caution: Cut the smallest possible gel slice. 4 Place the agarose gel slice into a sterile 1.5 ml microcentrifuge tube of known weight, then reweigh the tube to determine the weight of the agarose gel.
5 6 7 To the microcentrifuge tube, add 300 µl Binding Buffer for every 100 mg agarose gel. To dissolve the agarose gel slice and release the DNA: Vortex the microcentrifuge tube briefly to resuspend the gel slice in the Binding Buffer. Incubate the suspension for 10 min at 56 C. Vortex the tube briefly every 3 min during the incubation. After the gel is completely dissolved: To the microcentrifuge tube, add 150 µl isopropanol for every 100 mg agarose gel. Vortex thoroughly. 8 After mixing the components of the microcentrifuge tube: Insert one High Pure Filter Tube into one Collection Tube. Pipette the entire contents of the microcentrifuge tube into the upper Caution: If the volume of gel suspension is > 700 l, divide the suspension into two portions and use separate filter tubes for each portion. 9 Follow Protocol Va above, starting at the centrifugation step (Step 3). VI. Troubleshooting the High Pure protocols The same troubleshooting procedure can be applied to all High Pure kits. For details on how to troubleshoot the above protocols, see the General Troubleshooting Procedure for all High Pure kits on page 46 of this manual. For factors that may affect the High Pure PCR Product Purification Kit, see page 48. 3
Typical results with the kit Figure 5. Comparison of PCR products purified with the High Pure PCR Product Purification Kit and those purified by phenol/chloroform extraction. Four different long PCR products were generated with the Expand Long Template PCR System, purified by either of two methods, then analyzed electrophoretically. From left to right, the four products shown on the gel are: a.9 kb fragment from the p53 gene; 4.8 kb, 6.3 kb, and 9.3 kb fragments from the tissue plasminogen activator (tpa) gene. For each size product, the gel shows: Lane 1: DNA before purification; Lane : DNA after purification by phenol/chloroform extraction; Lane 3: DNA after purification with the High Pure PCR Product Purification Kit. Result: The High Pure Kit removed primers and primer dimers, while recovering 90% of the long PCR products (as calculated by photospectroscopy). Experiment 1..9 kb p53 4.8 kb tpa 6.3 kb tpa 9.3 kb tpa 1 3 1 3 1 3 1 3 Experiment. Preparation of PCR templates from primary fecal cultures for the detection of Escherichia coli Shiga-like toxin genes [from Dr. Udo Reischl and Birgit Haber, Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef- Strauss-Allee 11, 93053 Regensburg, Germany] Background: Escherichia coli strains producing one or more Shiga-like toxins (SLTs) can cause human diseases such as diarrhea, hemorrhagic colitis, and hemolyticuremic syndrome. Since the nucleotide sequence of the toxin genes are known, SLT genes may be detected by hybridization or by PCR. In routine diagnostic procedures, the samples to be screened for SLT genes are usually individual bacterial colonies from an overnight culture (on MacConkey agar) of a stool sample. However, fewer than 1% of E. coli isolates from such cultures may be SLT-positive, so the test may be relatively insensitive. This sensitivity limitation might be overcome if crude extracts of primary fecal cultures were directly screened by PCR. However, fecal samples contain potent DNA polymerase inhibitors (heme, bilirubin bile salts). Fecal DNA would have to be extensively purified before it could be used as a PCR template. While researching the screening of stool samples by PCR, this laboratory used the Boehringer Mannheim High Pure PCR Product Purification Kit to rapidly prepare PCR templates from primary fecal cultures. Methods: Purification of DNA from primary fecal cultures. Primary fecal cultures were prepared according to the method of Paton et al. (1993). For each culture, approximately 500 mg of feces was inoculated into 10 ml of Luria-Bertani (LB) broth and incubated at 37 C for at least 4 h. A 1 ml aliquot of each culture was centrifuged (14,000 rpm, 3 min). The pellet was resuspended in 95 µl of 0.1 M Tris-EDTA buffer (ph 8.0) and 5 µl of Proteinase K solution (0 mg/ml; Boehringer Mannheim) was added. After a 0 min incubation at 7 C, the sample was boiled for 10 min and centrifuged (14,000 rpm, 3 min). The clear supernatant (100 µl) was transferred to a new tube and 500 µl of Binding Buffer (from the High Pure PCR Product Purification Kit) was added. DNA was purified as described in Protocol Va above and used directly in PCR. Isolation of fecal DNA by standard procedures. For comparison, a separate 1 ml aliquot from each primary fecal culture was processed by centrifugation and Proteinase K treatment as described above. DNA in the clear supernatant from the Proteinase K digestion was precipitated with ethanol and used directly in PCR. 4
SLT-specific PCR. A 10 µl aliquot of each purified or isolated DNA was amplified in a 100 µl reaction mix containing dntps (00 µm each); primer SLT-1 and primer SLT- (1 µm each) (Paton et al., 1993); Taq DNA polymerase (1 unit; Boehringer Mannheim); and 10 µl of 10 x PCR buffer (Boehringer Mannheim). After an initial denaturation step (5 min, 94 C), samples were subjected to 35 PCR cycles [1 cycle = denaturation (1 min, 94 C), annealing (1 min, 60 C) and elongation (1 min, 7 C)]. Screening for specific amplification product. A quick screen for SLT bands was performed by electrophoresing an aliquot of each reaction mixture on a 1.5% agarose gel and staining the gel with ethidium bromide. In a more specific and sensitive analysis, a 0 µl aliquot of each reaction mixture was analyzed by an ELOSA (enzyme-linked oligosorbent assay) which used digoxigenin-labeled amplicons and biotin-labeled, SLT-specific probes. Results: Several primary cultures of SLTpositive stool specimens were screened by PCR. Figure 6 shows the PCR products obtained. Figure 6. SLT-specific PCR using DNA templates prepared from SLT-positive primary fecal cultures. Four stool specimens which were known to contain E. coli Shiga-like toxins (SLTs) were cultured. DNA from each primary fecal culture was either isolated by standard methods (proteinase K digestion, followed by ethanol precipitation) or purified to remove inhibitors of Taq DNA polymerase (proteinase K digestion, followed by purification with the ). Both isolated DNA and purified DNA were used as templates for PCR amplification with SLT-specific primers. (Details of all these procedures are given in the text.) The resultant PCR products from each of the four cultures are shown on this gel. Lanes 1 4: PCR product obtained when the template DNA from each culture was isolated by standard methods, without removal of polymerase inhibitors. Lanes 5 8: PCR product obtained when DNA from each culture was purified with the High Pure Kit to remove polymerase inhibitors. Lane 9: negative control (no DNA); Lane 10: positive control (DNA purified from SLT-positive E. coli strain O157:H7). MW, DNA Molecular Weight Marker VIII (Boehringer Mannheim). Result: Since the stool specimens contained DNA polymerase inhibitors, DNA samples prepared directly from the cultures by standard procedures (Proteinase K digestion, followed by ethanol precipitation) were not suited as PCR templates (lanes 1 4). However, when PCR templates from the same cultures were purified by the, the polymerase inhibitors were removed and each template produced a clearly visible, SLT-specific product (lanes 5 8). 5
Ordering information for the kit and related products Product Cat. No. Pack Size High Pure PCR Product 1 73 668 Purification Kit 1 1 73 676 Expand Long Template PCR System 1 681 834 1 681 84 1 759 060 50 reactions 50 reactions 100 units x 50 units 10 x 50 units Expand High Fidelity PCR System 1 73 641 1 73 650 1 759 078 100 units 500 units ( x 50 units) 500 units (10 x 50 units) PCR Core Kit 1 578 553 100 reactions PCR Nucleotide Mix 1 1 581 95 1 814 36 100 reactions 1000 units Taq DNA polymerase 3 (5 units/ l) 1 146 165 1 146 173 1 418 43 1 596 594 1 435 094 100 units 500 units 4 x 50 units 10 x 50 units 0 x 50 units (1 unit/ l) 1 647 679 1 647 687 Agarose LE 1 685 651 1 685 660 1 685 678 Agarose LM-MP 1 690 701 1 441 345 1 441 353 Agarose MP 1 444 964 1 388 983 1 388 991 Agarose MS 1 816 578 1 816 586 1 816 594 50 units 4 x 50 units 5 g 500 g 10 g 50 g 0 g 500 g 5 g 500 g Proteinase K, solution (14 mg/ml) 1 413 783 1 373 196 1 373 00 1.5 ml 5 ml 5 ml 1 This product is sold under licensing arrangements with Roche Molecular Systems and The Perkin-Elmer Corporation. For complete license disclaimer, see disclaimer 1, page II. This product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for life science research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. For complete license disclaimer, see disclaimer, page II. 3 This product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for life science research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. For complete license disclaimer, see disclaimer 3, page II. Reference Paton, A.W., Paton, J.C., Goldwater, P.N. and Manning, P.A. (1993) J. Clin. Microbiol. 31, 3063 3067. 6