1 Mag-Bind RX Plasmid 96 Kit Table of Contents Introduction...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4 Mag-Bind RX Plasmid 96 Protocol...5 Troubleshooting Guide...8 Manual Revision: February 2012 Innovations in nucleic acid isolation 1
2 Introduction The Mag-Bind family of products is an innovative system that radically simplifies extraction and purification of nucleic acids from a variety of sources. Key to the system is Omega Bio-Tek s proprietary Mag-Bind Particle that avidly, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or low salt buffer. The Mag-Bind RX Plasmid 96 Kit uses alkaline-sds lysis to generate the bacterial lysate and incorporates Mag-Bind magnetic particles for both lysate clearing and plasmid purification. By using magnetic particles to clear cell lysate, the Mag-Bind RX Plasmid 96 Kit can completely eliminate the need of vacuum or centrifugation, allowing DNA purification to be fully automated. Up to 96 samples can be simultaneously processed in less than 60 minutes. The purified plasmid can be used directly for automated fluorescent DNA sequencing, such as with BigDye sequencing chemistry, as well as for other standard molecular biology techniques including restriction enzyme digestion. New in this Edition: This manual has been edited for content and redesigned to enhance user readability. 2
3 Kit Contents Product M M M Purifications 1 x 96 4 x x 96 Mag-Bind Particles CND 1.1 ml 4.2 ml 25 ml Solution I 15 ml 60 ml 300 ml Solution II 15 ml 60 ml 300 ml MagN3 Buffer 15 ml 60 ml 300 ml SPM Wash Buffer 30 ml 120 ml 3 x 240 ml Elution Buffer 15 ml 60 ml 300 ml RNase A 75 μl 300 μl 1.8 ml User Manual P P P Storage and Stability All Mag-Bind RX Plasmid 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Solution I/RNase A, Mag-Bind Particles CND, and MagN3 Buffer should be stored at 2-8 C and all other components at C. HiBind, E.Z.N.A., and MicroElute are registered trademarks of Omega Bio-tek, Inc. PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license. 3
4 Preparing Reagents 1. Dilute SPM Wash Buffer with 100% ethanol as follows and store at room temperature. Kit 100% Ethanol to be Added M ml M ml M ml per bottle 2. Add the vial of RNase A to Solution I before use. Store at 2-8 C. 4
5 Mag-Bind RX Plasmid 96 Protocol Mag-Bind RX Plasmid 96 Kit Protocol Materials and Equipment to be Supplied by User: Centrifuge with swinging-bucket rotor capable of 3,000 x g Adapter for 96-well deep-well plates Magnetic Separation Device A (Cat# MSD-01) or Magnetic Separation Device B (Cat# MSD-01B) 500 μl Round-well Microplate (Cat# EZ /02) 0.5 ml Deep-well Plate (Cat# 9500 or Axygen P-DW-500-C)) Multichannel Pipettor Isopropanol 100% ethanol Before Starting: Prepare SPM Wash Buffer and Solution I according to the Preparing Reagents section on Page Innoculate ml LB/antibiotic(s) medium placed in a 96-well 2 ml culture plate and grow at 37 C with agitation for hours. Note: It is strongly recommended that an enda negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5α and JM Seal the plate with tape or sealing film. 3. Centrifuge at 3,000 x g for 15 minutes at room temperature. 4. Aspirate and discard supernatant. 5. Place the plate upside-down on a paper towel to remove excess media. 6. Add 120 μl Solution I to each sample. Shake or pipet to completely resuspend the cells. Note: Complete resuspension of cell pellet is vital for obtaining good plasmid yields. 5
6 Mag-Bind RX Plasmid 96 Protocol 7. Add 120 μl Solution II to each sample. Mix thoroughly by gently pipetting up and down 20 times. Note: Alternately, gently shake and rotate the plate for 1 minute to obtain a cleared lysate. A 5 minute incubation at room temperature may be necessary. Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.) 8. Add 120 μl MagN3 Buffer to each sample. Vortex or shake side to side to mix the lysate. Note: MagN3 Buffer contains magnetic beads and should be completely resuspended by vortexing before adding to the sample. 9. Place the plate on a magnetic separation device to magnetize the MagN3 magnetic beads. Let sit for minutes. 10. Transfer 230 μl cleared sample to a 500 μl round-well plate. Discard the 2 ml culture plate. 11. Add 10 μl Mag-Bind Particles CND and 80 μl isopropanol to each sample. Mix thoroughly by gently pipetting up and down 20 times. Note: The Mag-Bind Particles CND will settle and bead together at the bottom of the container after several hours. Mix thoroughly by vortexing before use. 12. Let sit for 3 minutes at room temperature. Mix once by pipetting. 13. Place the plate on a magnetic separation device to magnetize the Mag-Bind Particles CND. Let sit for 5 minutes or until the supernatant is clear. 14. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind Particles CND. 15. Remove the plate from the magnetic separation device. 6
7 Mag-Bind RX Plasmid 96 Protocol 16. Add 300 μl SPM Wash Buffer to each sample. Note: SPM Wash Buffer must be diluted with ethanol prior to use. Please see Page 4 for instructions. 17. Let sit for 40 seconds. Resuspend the Mag-Bind Particles CND by vortexing or pipetting up and down 20 times. 18. Place the plate on a magnetic separation device to magnetize the Mag-Bind Particles CND. Let sit for 5 minutes or until the supernatant is clear. 19. Aspirate and discard the cleared supernatant. Do not disturb the Mag-Bind Particles CND. 20. Repeat Steps twice for a second and third SPM wash step. 21. Leave the plate on the magnetic separation device for 5-10 minutes to air dry the Mag-Bind Particles CND. Remove any residual liquid with a pipettor. 22. Remove the plate from the magnetic separation device. 23. Add 100 µl Elution Buffer to each sample. Resuspend the Mag-Bind Particles CND by pipetting up and down 50 times or vortexing for 3 minutes. Note: Incubation at 37 C for 5-7 minutes may increase the yield. 24. Place the plate on a magnetic separation device to magnetize the Mag-Bind Particles CND. Let sit for minutes or until the supernatant is clear. 25. Transfer the cleared supernatant containing purified DNA to a clean plate. Store the DNA at -20 C. 7
8 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at Problem Cause Solution Low DNA yields No DNA eluted High-molecularweight DNA contamination Optical densities do not agree with DNA yield on agarose gel RNA visible on agarose gel DNA floats out of well while loading agarose gel Problem with downstream applications Poor cell lysis Bacterial culture overgrown Low copy-number plasmid used Lost Mag-Bind Particles CND during procedure SPM Wash Buffer not diluted with ethanol Over mixing of cell lysate upon addition of Solution II Trace contaminants eluted increase A 260 RNase A not added to Solution I Ethanol not completely removed before elution Ethanol carry over Do not use more than 6OD.600 bacterial culture. Cells may not be resuspended adequately after addition of Solution I. Vortex cells to completely disperse. Increase incubation time with Solution II to obtain a completely lysis. Solution II must be tightly closed when not in use. Solution II may need to be replaced. Do not incubate cultures for more than 20 hours at 37 C. Storage for extended periods prior to plasmid isolation is detrimental. Such plasmids may yield as little as 0.1 μg DNA from a 1 ml overnight culture. Carefully aspirate the supernatant during the procedure. Prepare SPM Wash Buffer as instructed on Page 4. Do not vortex or mix aggressively after adding Solution II. Gently shake and rotate the plate. Wash Mag-Bind Particles CND as instructed. Rely on agarose gel/ ethidium bromide electrophoresis for quantification. Add 1 vial of RNase to each bottle of Solution I. Increase dry time before elution step Make sure to remove all SPM Wash Buffer during the Mag-Bind Particles CND drying step.