Plasmid Midiprep Purification Kit Cat. # : DP01MD/ DP01MD-100 Size : 20/100 Reactions Store at RT For research use only 1
Description: The Plasmid Midiprep Purification Kit provides simple rapid protocol and materials to purify DNA from 20~100 ml bacteria liquid culture. The yield of DNA, from 30 μg~300 μg, is quantitative and reproducible. The obtained DNA can be used for many applications, including PCR, restriction enzyme digestion, cloning, automated DNA sequencing, southern blot, in vitro transcription assay, and transfection. Components of the Kit: DP01MD DP01MD-100 1. Solution I 110 ml 550 ml 2. Solution II 110 ml 550 ml 3. Solution III 110 ml 550 ml 4. Binding Solution 33 ml 165 ml 24 ml 120 ml 5. Endotoxin Removal (add 16 ml isopropanol (add 80 ml isopropanol Wash Solution before use) before use) 6. Wash Solution 10 ml (add 40 ml Ethanol before use) 50 ml (add 200 ml Ethanol before use) 7. Elution Solution 20 ml 100 ml 8. Midi-Spin Column 20 pcs 100 pcs 9. Collection Tube 20 pcs 100 pcs 10. RNase A powder 40 mg / btl. 180 mg x 1 +18 mg x 1 * Store RNase A Powder at -20 C and store all other kit components at room temperature (25~28 C). * If precipitates have formed in solution II, warm the buffer at 35 C in a water bath until fully dissolved. 2
Before staring this procedure: Dissolve RNaseA Powder in Solution I: For DP01MD, add 1 ml Solution I to each tube of RNase A Powder, vortex until completely dissolved. Transfer all the solution to Solution I and store at 4 C. For DP01MD-100, add one tube of 180 mg RNase A Powder to the 500 ml Solution I bottle, and add one tube of 18 mg RNase A Powder into the 50 ml Solution I bottle. General Procedure: * Use 20~50 ml of bacteria culture with high copy plasmid, and 50~100 ml of bacteria culture with low copy plasmid. Excess volume will result in incomplete cell lysis, reducing the yield and quality of plasmid. * Do not use sample volume more than recommended to prevent clogging of spin- column. 1. Pellet down cells from 20~100 ml liquid culture by centrifugation at 12,000~14,000 x g for 5 min. Pour off the supernatant. 2. Completely resuspend the cell pellet in 5 ml of Solution I by pipetting or vortexing. Incubate at RT for 10 min for uniform and complete suspension of cell especially for high density (> 3 x 10 9 cells/ml) or large volume (> 50 ml) cultures. 3. Add 5 ml of Solution II and mix by inverting the tube 6~8 times (the cell suspension should turn clear immediately). Incubate at RT for 2~4 min. * Incubation >5 min may promote partial denaturation of DNA and hence may affect manipulation like restriction digestion. 4. Add 5 ml of Solution III and mix it by inverting the tube 6~8 times. 5. Centrifuge the lysate at 12,000~14,000x g for 10 min at 4 C. A compact white pellet will form along the side or at the bottom of the tube. 6. Carefully transfer the supernatant to a new 50 ml centrifuge tube and 3
add 10 ml of isopropanol into the tube. Mix the solution by inverting the tube several times. A few of the floating white pellets may be transferred to the tube. This does not affect the purification of plasmid. 7. Centrifuge the lysate at 12,000~14,000x g for 10 minutes at 4 C. A white DNA pellet will form at the bottom of the tube. Discard the supernatant. 8. Add 500 μl of Binding Solution to resuspend the DNA pellet completely by pipetting gently. * Most DNA pellets will be at the bottom. For DNA pellets that are stuck to the wall of the tube, wash down and dissolve. 9. Insert the Midi-Spin Column into a Collection Tube and transfer the resuspended DNA solution to the Midi-Spin Column. Wait for 1 min for equilibration with the membrane and spin at top speed (12,000~14,000x g) for 2 min. 10. Discard the filtrate and add 500 μl of Binding Solution. Wait for 2 min for equilibration with the membrane and spin at top speed for 2 min. If endonuclease free is desired, add 500 μl binding solution to wash again. * This step eliminates the endonuclease contamination from EndA + strain (e.g. BL21 (DE3), HB101 and JM series (e.g. JM109). 11. Discard the filtrate and add 650 μl of Endotoxin Removal Wash Solution (with isopropanol added). Wait for 2 min for equilibration with the membrane and centrifuge at top speed for 2 min. Discard the filtrate. Repeat this step once more. 12. Discard the filtrate and add 600 μl of Wash Solution to the spin column. Centrifuge for 2 min and discard the filtrate. Repeat this step once more. * For transfection purpose, wash two more times, i.e. three washes in total. 13. Discard the filtrate and centrifuge for additional 5 min at top speed to remove residual ethanol. 14. Discard the collection tube and incubate the Midi-Spin Column at 60~65 C for 10 min to evaporate any residual ethanol. Do not incubate 4
the column > 15 min to prevent the DNA pellet from becoming too hard to elute. * Make sure to remove all ethanol completely, which may be important for automatic sequencing and transfection. 15. Transfer the Midi-Spin Column into a new 1.5 ml microcentrifuge tube and add 150~300 μl preheated (60~65 C) Elution Solution or TE or H 2 O (ph 7.0~8.5) into the column. Wait for 3 min and centrifuge at top speed for 2 min to elute DNA. Store the elute plasmid DNA at -20 C. *Notes: To increase the DNA concentration, reload the eluted DNA Solution into the Midi-Spin Column, and incubate at 60~65 C for 3 min and then spin again to elute DNA. 5
Troubleshooting Guide Low or no yield Comments and Suggestion a) Plasmid did not propagate Inoculate the liquid medium containing recommended antibiotic with single colony of bacterial cells from a freshly streaked plate and grow the culture under appropriate conditions. b) Overgrown bacteria Overgrowth of culture leads to lysis of cells and degradation of DNA. Do not grow cultures longer than 12~16 hours. c) Not enough bacterial cells Ensure optimum growth of culture (OD 600 > 1) by incubating the culture overnight at recommended temperature with vigorous shaking. d) Column clogged due to excess sample volume Do not use sample volume more than recommended to prevent clogging of spin- column. e) Low-copy-number plasmid When using low-copy-number plasmids, use larger culture volumes. Even with larger culture volumes, yields of low-copy-number plasmids will be lower than those of high-copy-number plasmids. f) Poor cell lysis 1) Excessive growth of bacteria. The culture 6
medium should contain recommended antibiotic. 2) Poor cell suspension. Ensure complete resuspension of bacterial pellet by votexing or pipetting before adding Solution II. g) Incomplete DNA Elution 1) If plasmid is larger than 7 Kb, use preheated Elution solution (60~65 C ) to elute. 2) DNA should be eluted only with a low-salt buffer [e.g., Elution solution (10 mm Tris Cl, ph 8.5) or water]. Elution efficiency is dependent on ph. The maximum efficiency is achieved between ph 7.0 and 8.5. When using water for elution, make sure that the ph value is within this range. 3) Ensure that Elution solution is added onto the center of the membrane and is completely absorbed. Genomic DNA contamination a) Overgrown bacteria Overgrowth of culture leads to lysis of cells and degradation of DNA. Do not grow cultures longer than 12~16 hours. b) Lysate prepared incorrectly 1) The lysate must be handled gently after addition of Solution II to prevent genomic 7
DNA shearing. 2) Lysis in step 3 must not exceed 5 minutes. c) Solution III added incorrectly Upon addition of Solution III, mix immediately, but gently. RNA contamination RNase A digestion insufficient 1) Ensure that RNase A is added to Solution I before use and stored at 4 C. 2) If Solution I containing RNase A is stored beyond a year, add additional RNase A to Solution I to make 360 μg/ml final concentration of RNase A. 3) Reduce the culture volume, if necessary. 8
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