BIO 101 Protocol w eb RPM Rapid Pure Miniprep Kit 2070-000 6 preps 2070-200 60 preps 2070-400 120 preps 2070-500 300 preps 2070-600 600 preps Rapid Isolation and Purification of Double- Stranded Plasmid DNA from Bacterial Cultures 12 preps in less than 12 Minutes GENECLEAN -Quality Pure Plasmid DNA for Cutting, Cloning, Sequencing, Amplification No Phenol/Chloroform Extractions Shipping & Storage: The RPM Kit is shipped and stored at ambient temperature. Check with BIO 101... We have you time in mind! Revision #: 2070-999-7C03P 1070 Joshua Way, Vista CA 92083, USA Tel: (760) 598-7299 Fax (760)598-0116 E-mail: technical@bio101.com Website: www.bio101.com For technical support call (800) 424-6101
2 RPM Protocol Kit Components:...3 2070-000 (6 preps)... 3 2070-200 (60 preps)...3 2070-400 (120 preps)...4 2070-500 (300 preps)...4 2070-600 (600 preps)...5 Introduction... 6 Protocol... 7 Notes... 9 Product Use Limitation & Warranty... 10 General Information...10 Call (800) 424-6101 for Technical Support
3 Kit Components: 2070-000 (6 preps) Name Volume Cat # Pre-Lysis Buffer 1.0 ml 2070-001 Alkaline Lysis Solution 1.0 ml 2070-002 Neutralizing Solution 1.0 ml 2070-003 GLASSMILK SpinBuffer * 1.8 ml 2070-004 Wash Solution Concentrate ** 2.0 ml 2070-005 SPIN Filters 6 2070-006 Catch Tubes 6 2070-007 CIRCLEGROW Sample 8 capsules 3000-101 *GLASSMILK SpinBuffer contains guanidine thiocyanate, use with proper precaution. **Add 2.0 ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use. 2070-200 (60 preps) Name Volume Cat # Pre-Lysis Buffer 4.0 ml 2070-201 Alkaline Lysis Solution 7.0 ml 2070-202 Neutralizing Solution 7.0 ml 2070-203 GLASSMILK SpinBuffer * 16 ml 2070-204 Wash Solution Concentrate ** 12 ml 2070-205 SPIN Filters 60 2070-206 Catch Tubes 60 2070-207 CIRCLEGROW Sample 8 capsules 3000-101 *GLASSMILK SpinBuffer contains guanidine thiocyanate, use with proper precaution. **Add 12 ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use. Call (800) 424-6101 for Ordering Information
4 2070-400 (120 preps) Name Volume Cat # Pre-Lysis Buffer 7.0 ml 2070-401 Alkaline Lysis Solution 15 ml 2070-402 Neutralizing Solution 15 ml 2070-403 GLASSMILK SpinBuffer * 35 ml 2070-404 Wash Solution Concentrate ** 24 ml 2070-405 SPIN Filters 120 2070-406 Catch Tubes 120 2070-407 CIRCLEGROW Sample 20 capsules 3000-011 *GLASSMILK Spinbuffer contains guanidine thiocyanate, use with proper precaution. **Add 24 ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use. 2070-500 (300 preps) Name Volume Cat # Pre-Lysis Buffer 18 ml 2070-501 Alkaline Lysis Solution 33 ml 2070-502 Neutralizing Solution 33 ml 2070-503 GLASSMILK SpinBuffer * 90 ml 2070-504 Wash Solution Concentrate ** 60 ml 2070-505 SPIN Filters 300 2070-506 Catch Tubes 300 2070-507 CIRCLEGROW Sample 20 capsules 3000-011 *GLASSMILK Spinbuffer contains guanidine thiocyanate, use with proper precaution. **Add 60ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use. Call (800) 424-6101 for Technical Support
5 2070-600 (600 preps) Name Volume Cat # Pre-Lysis Buffer 35 ml 2070-601 Alkaline Lysis Solution 65 ml 2070-602 Neutralizing Solution 65 ml 2070-603 GLASSMILK SpinBuffer * 180 ml 2070-604 Wash Solution Concentrate ** 120 ml 2070-605 SPIN Filters 5 x 120 2070-606 Catch Tubes 5 x 120 2070-607 CIRCLEGROW Sample 40 capsules 3000-001 *GLASSMILK SpinBuffer contains guanidine thiocyanate, use with proper precaution. **Add 120ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use. All references to user supplied reagents are italicized and bolded in the protocol for your convenience. Call (800) 424-6101 for Ordering Information
6 Introduction The RPM Kit delivers up to 20 µg of supercoiled plasmid DNA with minimum effort and time. Multiple samples can be processed rapidly by using 1.5 ml of bacterial culture. With practice, the procedure can be completed in less than 12 minutes, yielding pure plasmid DNA suitable for enzymatic manipulations including DNA sequencing, restriction enzyme digestion, in vitro transcription 1, and PCR 2. Reagents and materials supplied by the user are 100% ethanol, sterile H 2 O or TE buffer, 1.5 ml microcentrifuge vials (one for each miniprep), and a microcentrifuge. The reagents and materials provided with the kit are for research use only. 1 For use of the plasmid DNA for in vitro transcription experiments, addition of RNase Inhibitor is strongly recommended. 2 PCR Process is covered by U.S. Patent Numbers 4.683.202 and 4.683.195 issued to Hoffman & LaRoche Corporation. Call (800) 424-6101 for Technical Support
7 Protocol 1. Spin 1.5 ml of bacterial culture (use Circlegrow media to grow cells to high density for maximum yield of plasmid DNA) in a microcentrifuge vial for 30 seconds, remove and discard supernatant. If use of more than 1.5 ml culture for this miniprep is desired, add more culture, re-spin, remove and discard supernatant. (Pour off supernatant and then remove the remaining few microliters with a pipet tip.) 2. To resuspend the cell pellet, add 50 µl Pre-Lysis Buffer and mix by vortexing or by pipetting up and down until completely suspended. 3. Add 100 µl Alkaline Lysis Solution directly into the cell suspension and mix by inverting the tube several times. Before proceeding to step 4, wait 1 minute to allow lysis to complete (solution should be clear). This is a critical step in the procedure and affects yield and purity of the plasmid DNA; the cells must be homogeneously lysed and some host cells lyse easier than others. For optimum consistency of lysis from one cell type to another, be certain the Alkaline Lysis Solution is 22-25 C and that the SDS is in solution. 4. Add 100 µl Neutralizing Solution and mix by briefly vortexing. A white precipitate will form consisting of cell membranes, proteins, and chromosomal DNA. Spin in a microcentrifuge for 2 minutes to pellet white precipitate. 4a. Prepare a SPIN Filter for each sample by adding 250 µl GLASSMILK SpinBuffer (mix suspension completely before each use) to the SPIN Filter chamber and placing it in a Catch Tube. 4b. Remove supernatant from sample while avoiding white pellet, and transfer to a prepared SPIN Filter (see step 4a). Call (800) 424-6101 for Ordering Information
8 5. Pipet up and down a couple of times to mix and, after 1 minute, spin for 1 minute to collect liquid in the bottom of the Catch Tube. 6. Add 350 µl Wash Solution (remember to add an equal volume of ethanol before first use)* to SPIN Filter and spin for 1 minute. Pour off liquid in collection vial and spin again for 1 minute to drive last of the liquid out of the SPIN Filter. Transfer SPIN Filter to a new Catch Tube. Discard other tube. 7. To elute plasmid DNA from the SPIN Filter, add 50 µl of autoclaved sterile H 2 O or TE buffer (10 mm Tris-HCl, ph 7.5, 1 mm EDTA), vortex briefly (no more than half speed) to resuspend GLASSMILK, and spin for 30 seconds to collect DNA in the bottom of the Catch Tube. Remove Catch Tube from microcentrifuge; remove and discard SPIN Filter. Close Catch Tube containing plasmid DNA and store at +4 C or -20 C. Note: The yield of plasmid DNA is dependent on the bacterial host and the plasmid copy number. The plasmid DNA is now ready for further manipulation. * Catalog # Description Ethanol Wash Sol. Final to add* Conc. Volume 2070-005 RPM Sample 2 ml 2 ml 4 ml 2070-205 RPM-60 12 ml 12 ml 24 ml 2070-405 RPM-120 24 ml 24 ml 48 ml 2070-505 RPM-300 60 ml 60 ml 120 ml 2070-605 RPM-600 120 ml 120 ml 240 ml * 100% Ethanol is provided by the user. Call (800) 424-6101 for Technical Support
9 Notes Call (800) 424-6101 for Ordering Information
10 Notes Call (800) 424-6101 for Technical Support
11 Notes Call (800) 424-6101 for Ordering Information
Product Use Limitation & Warranty Unless otherwise indicated, this product is for research use only. Purchase of BIO 101 products does not grant rights to reproduce, modify, repackage the products or any derivative thereof to third parties. BIO 101 makes no warranty of any kind, expressed or implied, including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer's exclusive remedy and BIO 101's sole liability hereunder shall be limited to, at our option, product credits, refund of the purchase price of, or the replacement of all material(s) that does not meet our specification. By acceptance of the product, Buyer indemnifies and holds BIO 101 harmless against, and assumes all liability for the consequence of its use or misuse by the Buyer, its employees or others, including, but not limited to, the cost of handling. Said refunds or replacement is conditioned on Buyer notifying BIO 101 within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (days) shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). General Information BIO 101 is a pioneer in developing kits for molecular biology research. We introduced the GENECLEAN Kits in 1986 and have since been manufacturing products to bring convenience into your research. Our goal is to make your life easier by simplifying the complexities of lab work. Technical Support and Ordering Information (800) 424-6101 (Toll Free - Continental USA) (760) 598-7299 (Outside USA) (760) 598-0116 (Fax line) technical@bio101.com order@bio101.com http://www.bio101.com Office Hours: 6:30 am - 6:00 pm P.S.T. (Mon - Fri) Mailing Address: BIO 101, Inc. P.O. Box 2284 La Jolla, CA 92038-2284 USA