UltraClean Midi Plasmid Prep Kit

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1 UltraClean Midi Plasmid Prep Kit Catalog No. Quantity Preps Instruction Manual Please recycle Version:

2 Table of Contents Introduction... 3 Protocol Overview... 3 Flow Chart... 4 Equipment Required... 5 Kit Contents & Storage... 5 Precautions & Warnings... 5 Protocols: Short Protocol... 6 Detailed Protocol (Describes what is happening at each step)... 7 Hints & Troubleshooting Guide... 9 Contact Information Products recommended for you

3 Introduction The UltraClean Midi Plasmid Prep Kit is designed to isolate plasmid DNA from of E. coli. This kit uses the same silica spin filter technology employed in the UltraClean 6 Minute Mini Plasmid Prep Kit. Unlike other kits, this midi plasmid prep kit can be used with LB Broth culture as well as high nutrient broth culture media including Terrific Broth, TB DRY, and 2X YT. The recommended starting volume is 50 ml of culture. Protocol Overview The procedure is to lyse cells with an improved set of alkaline lysis reagents. Plasmid DNA is bound to a silica spin filter, washed once and recovered in Tris elution buffer. The DNA is concentrated enough to be used directly in automated sequencing. The DNA is also ready for restriction digests, labeling, PCR and other downstream applications. This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity TB DRY Powder Growth Media g 1 kg 5 kg UltraClean Maxi Plasmid Prep Kit preps 20 preps UltraClean 6 Minute Mini Plasmid Prep Kit preps 100 preps 250 preps 100 bp DNA Ladder µg 3

4 4

5 Equipment Required Vortex-Genie 2 Vortex (MO BIO Catalog# V or V-220) Centrifuge capable of spinning 15 ml and 50 ml tubes at 4500 x g (swinging bucket rotor is recommended) Note: The maximum g-force rating of the 15 ml Collection Tubes is 7,100 x g and for the 50 ml Centrifuge Tubes is 9,000 x g. Do not exceed 6,000 x g in a 50 ml Centrifuge Tube filled with 50 ml of culture in the tubes provided. Kit Contents Kit Catalog# Component Catalog# Amount Solution ml Solution ml Solution ml Solution x 30 ml Solution ml Spin Filters SF ml Collection Tubes T ml Centrifuge Tubes T ml Collection Tubes T3 20 Kit Storage Kit reagents and components should be stored at room temperature (15-30 C). Precautions Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact, wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact. All MSDS information is available upon request ( ) or at Reagents labeled flammable should be kept away from open flames and sparks. Note: All centrifugation steps in the 15 ml Collection Tubes are done without a cap. WARNING: Solution 4 is flammable. Do not let Solution 3 come into contact with bleach or other oxidizers. 5

6 Short Protocol Please wear gloves at all times 1. In a 50 ml Centrifuge Tube (provided) centrifuge 50 ml of culture at 4500 x g for 10 minutes. 2. Discard supernatant, spin again briefly and remove the remaining volume with a pipet tip. 3. Add 0.9 ml Solution Resuspend the bacterial pellet by bump vortexing until homogeneous. 5. Check Solution 2 before use. If a precipitate has formed, heat to dissolve. Cool to room temperature (15-30 C) before using. 6. Add 1.8 ml Solution 2 and gently invert up to 8 times to mix. 7. Add 3.6 ml Solution 3 and gently invert up to 8 times to mix. 8. Centrifuge at 4500 x g for 15 minutes. 9. Place a Spin Filter in a 15 ml Collection Tube (provided). 10. Transfer 3.5 ml of the lysate supernatant to the Spin Filter (avoid the white precipitate). 11. Centrifuge (uncapped) for 3 minutes at 4500 x g. Warning: Do not expose the liquid flow through to bleach. 12. Discard the flow through then replace the Spin Filter unit in the 15 ml Collection Tube. 13. Add the remaining lysate to the Spin Filter and centrifuge (uncapped) for 3 minutes at 4500 x g. 14. Discard the flow through then replace the Spin Filter unit in the 15 ml Collection Tube. 15. Add 3 ml Solution 4 to the Spin Filter. 16. Centrifuge 3 minutes at 4500 x g. 17. Discard the flow through then replace the Spin Filter unit in the 15 ml Collection Tube and spin again for 5 minutes at 4500 x g. 18. Carefully place Spin Filter unit in a new 15 ml Collection Tube (provided) without splashing any liquid on the Spin Filter. 19. Allow to air dry for 10 minutes at room temperature. 20. Add 1 ml Solution 5 to the middle of the Spin Filter. 21. Centrifuge for 3 minutes at 4500 x g. 22. Remove Spin Filter, transfer the eluate to a 2 ml Collection Tube (provided) and close tube lid. Plasmid DNA is now ready to use for any application. Thank you for choosing the UltraClean Midi Plasmid Prep Kit. 6

7 Detailed Protocol (Describes what is happening at each step) Please wear gloves at all times 1. In a 50 ml Centrifuge Tube (provided), centrifuge up to 50 ml of an overnight culture for 10 minutes at 4500 x g. For maximum yields, the supernatant should be clear and the bacteria should form a tight pellet. If the supernatant is not clear, centrifuge longer or at a higher g force. What s happening: The bacterial cells are being forced to the bottom of the tube. 2. Discard the supernatant. IMPORTANT: Remove all liquid. Spin again briefly and remove the remaining volume with a pipet tip. What s happening: The cells have been pelleted and are now separated from the culture growth medium. Removing all remaining liquid insures that reagents will not be diluted by residual liquid growth media. 3. Add 0.9 ml Solution 1 and resuspend the bacterial pellet by vortexing until homogeneous (be sure no clumps exist). Resuspend the bacterial pellet by bump vortexing with the vortex set at the highest speed. Bump vortexing means: hold the tube tip on the vortex head for 10 seconds, take it off for 1 second then hold it on the vortex again. Repeat this process for 1 minute. After 1 minute, hold the tube in a horizontal position up to a light and look at it. The liquid will spread from one end of the tube to the other. If you see any clumps of cells, keep bump vortexing until they are gone. It takes a minimum of 1 minute with the vortex at its highest speed to resuspend cells. What s happening: The bacterial cells are resuspended in a small volume of buffer that keeps them from breaking open (lysing). It is important to make this suspension of cells homogeneous because cells trapped in clumps will be resistant to lysis reagents. Solution 1 contains RNase A; however, it cannot digest RNA until the cells are lysed in the next step. 4. Check Solution 2 before use. If a precipitate has formed, heat to dissolve. Cool to room temperature (15-30 C) before using. What s happening: Solution 2 contains a detergent SDS that can precipitate if cooled. This precipitate is easy to resuspend by heating. For this reason, always store this kit at room temperature (15-30 C). 5. Add 1.8 ml Solution 2 and gently invert up to 8 times to mix. What s happening: Solution 2 is very alkaline (ph 12) and contains the detergent SDS. Addition of Solution 2 causes the bacterial cells to lyse because the proteins in the cell membrane become denatured. All DNA becomes denatured to its single stranded form at this point. The bacterial chromosomal DNA is long and is attached to broken pieces of the cell membrane. Plasmid DNA is linked so it forms two attached circles. All RNA is digested during this very short step because RNase A is active even in very alkaline conditions. 6. Add 3.6 ml Solution 3 and gently invert up to 8 times to mix. What s happening: Solution 3 contains potassium acetate and salt. The potassium acetate forms a precipitate when it interacts with SDS. At the same time denatured proteins co-precipitate with the SDS. Solution 3 neutralizes the alkaline ph to a more neutral ph 7. All DNA tries to re-nature. Plasmid can easily re-anneal to its double stranded form. Bacterial chromosomal DNA has difficulty re-naturing because it has no reference point and homologous pieces of DNA may be blocked from finding each other by the cell debris present. 7. Centrifuge at 4500 x g for 15 minutes. If your centrifuge cannot attain this force, 2500 x g for 27 minutes is sufficient. 7

8 What s happening: Dense cell debris is pelleted to the bottom of the tube. Chromosomal DNA is also pelleted along with the cell debris. 8. Place a Spin Filter in a 15 ml Collection Tube (provided). 9. Transfer 3.5 ml of the liquid lysate supernatant to the Spin Filter (avoid the white precipitate when transferring the supernatant). The best way to do this is to keep the white pellet on the up side of the tube as you pour the liquid into the Spin Filter. If the white pellet does not appear tight enough to pour out the liquid, it may be necessary to centrifuge again. Sometimes there is a floating white material. Try to avoid pouring this into the Spin Filter. It can be held back with a pipet if necessary. 10. Centrifuge (uncapped) for 3 minutes at 4500 x g. Warning: Do not expose the liquid that flows through the Spin Filter and into the 15 ml Collection Tube to bleach. What s happening: The plasmid DNA binds to the white silica membrane in the Spin Filter. Plasmid DNA binds due to the high salt conditions. Unwanted impurities such as digested RNA, and any other cell components are passed through the Spin Filter and end up in the flow through in the Collection Tube. Flow through is discarded. 11. Remove the Spin Filter unit, discard the liquid contents from the 15 ml Collection Tube in a suitable container and then replace the Spin Filter unit in the 15 ml Collection Tube. 12. Add the remaining lysate to the Spin Filter and centrifuge (uncapped) for 3 minutes at 4500 x g. 13. Remove the Spin Filter unit, discard the liquid contents from the 15 ml Collection Tube in a suitable container and then replace the Spin Filter unit in the 15 ml Collection Tube. 14. Add 3 ml Solution 4 to the Spin Filter. What s happening: Solution 4 washes the DNA that is bound to the Spin Filter. The ethanol keeps the plasmid DNA bound to the filter as impurities are washed away. 15. Centrifuge 3 minutes at 4500 x g. 16. Discard the flow through then replace the Spin Filter unit in the 15 ml Collection Tube and spin again for 5 minutes at 4500 x g. (This will insure that no residual Solution 4 will be left on the Spin Filter.) 17. Carefully place Spin Filter unit in a new 15 ml Collection Tube (provided) without splashing any liquid on the Spin Filter as it is removed. Allow to air dry for 10 minutes at room temperature. 18. Add 1 ml Solution 5 to the middle of the Spin Filter. What s happening: Solution 5 is 10mM Tris. As it passes through the Spin Filter, the plasmid DNA is released (eluted) off the filter and into the Collection Tube. The plasmid DNA is released because it will not stay bound to the Spin Filter when there is no salt present. 19. Centrifuge for 3 minutes at 4500 x g. 20. Remove Spin Filter, transfer the eluate to a 2 ml Collection Tube (provided) and close tube lid. Plasmid DNA is now ready to use for any application. Thank you for choosing the UltraClean Midi Plasmid Prep Kit. 8

9 Hints and Troubleshooting Guide Concentrating the DNA Your DNA will be in a final volume of 1 ml in 10 mm Tris. If this is too dilute for your purposes, ethanol precipitate the DNA to concentrate it. Add 1/10 volume of 3 M potassium acetate and 2 volumes 100% cold ethanol (or 0.7 volumes of isopropanol at room temperature). Chill at -20 C for an hour or overnight. Centrifuge for 20 minutes at full speed of your microcentrifuge or if using a 15 ml tube, centrifuge for 30 minutes at 5000 x g. Discard the supernatant. Wash the pellet with 70% ethanol to remove excess salt. Centrifuge for 10 minutes at full speed to re-attach the pellet to the wall. Note the position of the pellet. Drain the tube on a paper towel for 5 minutes and then allow the pellet to air dry or use a speed vac. Resuspend the pellet in the desired volume of Solution 5. Amount of Culture to Process This kit is designed for 50 ml of culture. To achieve higher yields of plasmid DNA, use TB DRY Powder Growth Media (a one powder form of Terrific Broth manufactured by MO BIO Laboratories, Inc.). White Pellet After Solution 3 Neutralizing Step is Loose If you do not get a tight white pellet after adding Solution 3 and centrifuging but rather a loose pellet or flocculent solution, you may have left too much residual culture media in the tube after spinning down the cells. You will need to start over and be careful to remove all traces of liquid culture media after pelleting the cells. DNA Floats Out of Well When Loaded on a Gel Make sure to perform the 10 minute air dry step before elution to allow residual ethanol to evaporate completely. Another cause may be from inadvertent transfer of residual Solution 4 into the final sample. Prevent this by being careful not to transfer Solution 4 flow through onto the bottom of the Spin Filter basket prior to the elution step with Solution 5. If you suspect you have residual Solution 4 in your sample, ethanol precipitation is the best way to remove residues of Solution 4. See Concentrating the DNA above. Low Recovery Low plasmid recovery may be caused by several factors. The culture conditions are important for obtaining maximal yields. Prepare a starter culture in 5 ml from a fresh colony streaked on an antibiotic containing plate and grow for 8 hours. Inoculate the large culture containing fresh antibiotic with a 1:1000 dilution of the 8 hour starter culture and allow growth for hours. Allowing the culture to grow longer or inoculation with too much starter culture may result in the plasmid culture entering death phase before 12 hours, resulting in loss of plasmid due to autolysis. In addition, the antibiotic will be utilized quickly and loss of plasmid selection can occur. A culture in death phase will also result in increased genomic DNA contamination of the plasmid DNA. Inoculation of the larger culture with a colony from a plate or with an old culture may result in a long lag phase and harvest of the culture at a low density. It may also result in co-inoculation of the culture with non-plasmid containing satellite colonies that outgrow the plasmid containing bacteria if the antibiotic is not fresh. For optimal aeration during growth, the volume of the growth vessel should be at least four times more than the culture volume. 9

10 Contact Information Technical Support: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA Ordering Information: Direct: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA For the distributor nearest you, visit our web site at 10

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