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1 BIO 101 Protocol w eb RPM Rapid Pure Miniprep Kit preps preps preps preps 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 We have you time in mind! Revision #: C03P 1070 Joshua Way, Vista CA 92083, USA Tel: (760) Fax (760) Website: For technical support call (800)

2 2 RPM Protocol Kit Components: (6 preps) (60 preps) (120 preps) (300 preps) (600 preps)...5 Introduction... 6 Protocol... 7 Notes... 9 Product Use Limitation & Warranty General Information...10 Call (800) for Technical Support

3 3 Kit Components: (6 preps) Name Volume Cat # Pre-Lysis Buffer 1.0 ml Alkaline Lysis Solution 1.0 ml Neutralizing Solution 1.0 ml GLASSMILK SpinBuffer * 1.8 ml Wash Solution Concentrate ** 2.0 ml SPIN Filters Catch Tubes CIRCLEGROW Sample 8 capsules *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 (60 preps) Name Volume Cat # Pre-Lysis Buffer 4.0 ml Alkaline Lysis Solution 7.0 ml Neutralizing Solution 7.0 ml GLASSMILK SpinBuffer * 16 ml Wash Solution Concentrate ** 12 ml SPIN Filters Catch Tubes CIRCLEGROW Sample 8 capsules *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) for Ordering Information

4 (120 preps) Name Volume Cat # Pre-Lysis Buffer 7.0 ml Alkaline Lysis Solution 15 ml Neutralizing Solution 15 ml GLASSMILK SpinBuffer * 35 ml Wash Solution Concentrate ** 24 ml SPIN Filters Catch Tubes CIRCLEGROW Sample 20 capsules *GLASSMILK Spinbuffer contains guanidine thiocyanate, use with proper precaution. **Add 24 ml of 100% Ethanol to Wash Solution Concentrate and mix before initial use (300 preps) Name Volume Cat # Pre-Lysis Buffer 18 ml Alkaline Lysis Solution 33 ml Neutralizing Solution 33 ml GLASSMILK SpinBuffer * 90 ml Wash Solution Concentrate ** 60 ml SPIN Filters Catch Tubes CIRCLEGROW Sample 20 capsules *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) for Technical Support

5 (600 preps) Name Volume Cat # Pre-Lysis Buffer 35 ml Alkaline Lysis Solution 65 ml Neutralizing Solution 65 ml GLASSMILK SpinBuffer * 180 ml Wash Solution Concentrate ** 120 ml SPIN Filters 5 x Catch Tubes 5 x CIRCLEGROW Sample 40 capsules *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) for Ordering Information

6 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 and issued to Hoffman & LaRoche Corporation. Call (800) for Technical Support

7 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 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) for Ordering Information

8 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 RPM Sample 2 ml 2 ml 4 ml RPM ml 12 ml 24 ml RPM ml 24 ml 48 ml RPM ml 60 ml 120 ml RPM ml 120 ml 240 ml * 100% Ethanol is provided by the user. Call (800) for Technical Support

9 9 Notes Call (800) for Ordering Information

10 10 Notes Call (800) for Technical Support

11 11 Notes Call (800) for Ordering Information

12 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) (Toll Free - Continental USA) (760) (Outside USA) (760) (Fax line) 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 USA

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