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Download M1205-2, -4 96 Well Gel Filtration Plate
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Transcript
FOR RESEARCH USE ONLY! 02/17 96 Well Gel Filtration Plate (Cat# M1205-2, -4; 2 or 4 Plates; Store at 4ºC) I. Introduction: The 96 Well Gel Filtration Plates are pre-packed with a highly efficient gel filter which is ideal for removing excess unincorporated dye terminators, freeing nucleotides from sequencing and labeling reactions, protein purification, desalting and buffer exchange. Low molecular weight material is retained in the gel matrix, facilitating the purification of DNA fragments larger than 20 bases in length. Since 96-Well G50 Gel Filtration Plates are designed to purify DNA fragments >20 bases only, they are not recommended for PCR product primer removal. II. Application: Removing excess unincorporated dye terminators Freeing nucleotides from sequencing and labeling reactions Protein purification Desalting Buffer exchange III. Key Features: Purification in only 10 minutes! Sample Volume: 20 to 50 μl Filtrate: >20 bases Filtration Plates are pre-packed with a highly efficient gel filter IV. Sample Type: DNA fragments larger than 20 bases in length V. Kit Contents (96 Well Gel Filtration Plate): Components M1209-2 M1209-4 96 Well Gel Filtration Plate 2 Plates 4 Plates VI. User Supplied Reagents and Equipment: 2 ml collection plate 0.35 ml collection plate VII. Shipment and Storage: All the reagents are shipped and stored at room temperature (4ºC) for up to atleast 6 months without showing any reduction in performance. VIII. Reagent Preparation and Storage Conditions: In the event of gel drying/cracking add 50-100 µl of ddH2O to each well of the G-50 Plate prior to use. IX. Purification/Removal/Desalting Protocol: Please read the entire instruction manual prior to starting the Protocol Procedure. 1. Plate Preparation Remove the adhesive film from the G-50 Plate. Place the G-50 Plate on a 2 ml collection plate. Centrifuge at 2,500g for 5 minutes. 2. Sample Loading Transfer the G-50 Plate to a 0.35 ml collection plate. Carefully load the sample (20-50 µl) onto the center of each gel filter surface. 3. Purification Recovery Centrifuge at 2,500g for 5 minutes. Each purified sample can be recovered at the bottom of the 0.35 ml collection plate. (approximately the same volume as the loaded sample). Buffer Exchange Protocol 1. Plate Preparation Remove the adhesive film from the G-50 Plate. Place the G-50 Plate on a 2 ml collection plate and centrifuge at 2,500g for 5 minutes. Discard the flow-through and place the G-50 Plate back on the same 2 ml collection plate. 2. Buffer Loading Add 350 µl of desired buffer to each well of the G-50 Plate. Centrifuge at 2,500g for 5 minutes. 3. Sample Loading Transfer the G-50 Plate to a 0.35 ml collection plate. Carefully load the sample (20-50 µl) onto the center of each gel filter surface. 4. Purification Recovery Centrifuge at 2,500g for 5 minutes. Each purified sample can be recovered at the bottom of the 0.35 ml collection plate (approximately the same volume as the loaded sample). X. Functional Test Data: Test Original DNA 2 3 DNA Conc 23.70 ng/µl 18.7 ng/µl 16.2 ng/µl Total DNA 1185 ng 935 ng 810 ng Recovery ----------78.9 % 87.3% Figure 1. Gel slice DNA fragments ranging from 250 bp-10 kb were extracted using the Gel Extraction Kit (lane 1,2,3). The purified DNA from a 50 µl eluate was analyzed by electrophoresis on a 1% agarose gel. M = 1 Kb DNA Ladder (control, total DNA = 1100 ng). 155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected] FOR RESEARCH USE ONLY! XI. 02/17 Related Products: Product Name Avg DNA Frag Extraction Kit DNA Cleanup Kit DNA Cleanup Midi Kit DNA Cleanup Maxi Kit Ext Gel/PCR Combo Kit Gel Extraction Kit Gel Filtration Columns Gel Filt. Dye Removal Columns Mini DNA Frag Extraction Kit 96 Well Gel Extraction Kit 96 Well Gel Filtration Plate 96 Well PCR Cleanup Kit PCR Cleanup Binding Plate PCR Cleanup Kit Trad Gel/PCR Combo Kit XII. Cat. No. K1366-100, -300 K1367-100, -300 K1368-100, -300 K1369-10, -25 K1364-100, -300 K1362-100, -300 M1206-2, -50 M1207-2, -50 K1367-100, -300 K1370-2, -4 M1205-2, -4 K1371-2, -4 M1208-5 K1365-100, -300 K1363-100, -300 Size 100 Preps, 300 Preps 100 Preps, 300 Preps 100 Preps, 300 Preps 10 Preps, 25 Preps 100 Preps, 300 Preps 100 Preps, 300 Preps 2 Pcs, 50 Pcs 2 Pcs, 50 Pcs 100 Preps, 300 Preps 2 Plates, 4 Plates 2 Pcs, 4 Pcs 2 Plates, 4 Plates 5 Plates 100 Preps, 300 Preps 100 Preps, 300 Preps General Troubleshooting Guide: Problem Low RNA Yield Cause . Agarose gel did not dissolve completely . Incomplete Wash Buffer preparation. . Incorrect DNA elution step Eluted DNA Does Not Perform Well in Downstream Applications . DNA was denatured (a smaller band appeared on gel analysis). . Primer dimer contamination in the final PCR elution product. Solution . Ensure the agarose gel was melted/dissolved completely between 55-60ºC for 10-15 minutes, or until no gel is visible. If undissolved agarose remains in the sample, the DFH Column could clog and some DNA will be unrecoverable. DNA can be denatured if the incubation temperature exceeds 60ºC. If using more than 300 mg of agarose gel, separate it into multiple 1.5 ml microcentrifuge tubes. . Add absolute ethanol (see the bottle label for volume) to Wash Buffer then mix by shaking for a few seconds. Check the box on the bottle. Be sure and close the bottle tightly after each use to avoid ethanol evaporation. . Ensure that Elution Buffer, TE or water is added into the CENTER of the DFH Column matrix and is completely absorbed. If DNA fragments are larger than 5 kb, use preheated Elution Buffer, TE, or water (60~70ºC). If using water for elution, ensure the water pH is ≥8.0. ddH2O should be fresh as ambient CO2 can quickly cause acidification. . Ensure the agarose gel was melted/dissolved completely between 55-60ºC for 10-15 min, or until no gel is visible. DNA can be denatured if the incubation temperature exceeds 60ºC. Incubate the eluted DNA at 95ºC for 2 minutes then cool down slowly to reanneal the denatured DNA. . Gel purification should be performed if primer dimers are visible in the agarose gel following PCR reactions. Simply cut the PCR product from the gel and avoid the primer dimer. Using an additional 80% ethanol wash will reduce primer dimer contamination when performing PCR cleanup. FOR RESEARCH USE ONLY! Not to be used on humans. 155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected]