Plasmid Midiprep Plus Purification Kit Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only 1
Description: The Plasmid Midiprep Plus Purification Kit provides simple rapid protocol and materials to purify plasmid DNA from 50~200 ml of liquid bacterial culture. The yield of plasmid DNA, from 100 μg~500 μg, is quantitative and reproducible, and can be used for most subsequent applications, including PCR, restriction enzyme digestion, cloning, automated DNA sequencing, southern blot, in vitro transcription assay, and transfection. Components of the Kit: DP01MD-P DP01MD-P50 1. Solution I 55 ml 280 ml 2. Solution II 55 ml 280 ml 3. Solution III 55 ml 280 ml 4. Endotoxin Removal 72 ml 360 ml Wash Solution (add 48 ml isopropanol (add 240 ml isopropanol before use) 5. Wash Solution 50 ml (add 200 ml Ethanol before use) before use) 120 ml x 2 (add 480 ml Ethanol before use) 6. Elution Solution 30 ml 150 ml 7. Midi-Spin Column 10 pcs 10 pcs x 5 8. 50 ml Collection Tube 10 pcs 10 pcs x 5 9. RNase A powder 20 mg 100 mg 10. Filter Net 10 pcs 10 pcs x 5 * Store RNase A Powder at -20 C and store all other kit components at RT. *If precipitates have formed in solution II, warm the buffer at 35 C in a water bath until fully dissolved. 1
Things to do before starting: Dissolve RNase A Powder in Solution I: Add 1 ml Solution I to each tube of RNase A Powder, vortex to completely dissolve, transfer all the solution to the Solution I and store at 4 C. Add isopropanol [48 ml for DP01MD-P10 (or) 240 ml for DP01MD-P50] to Endotoxin Removal Wash Solution and mix well. Store at RT. Add ethanol [200 ml for DP01MD-P10 (or) 480 ml for DP01MD-P50] to Wash Solution and mix well. Store at RT. Turn on water bath at 65~70. General Procedure: * Use 50~100 ml of bacterial culture with high copy plasmids, and 100~200 ml for bacteria culture with low copy plasmids. Excess volume can 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 50~200 ml liquid culture at 4,000 x g for 10 min. Pour off the supernatant. 2. Completely resuspend the cell pellet in 5 ml of Solution I by pipetting. 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 x g for 10 minutes at 4 C. A compact white pellet will form along the side or at the bottom of the tube. 2
6. To remove the floating pellet, hold a Filter Net by hand above a sterile 50 ml centrifuge tube (not provided) and gently pass the lysate through the filter net. See the figure below. 7. Open the cap of a Midi-Spin Column provided in 50 ml centrifuge tube and add about 12 ml (< 15 ml) of cleared lysate into the column. Close the cap and wait for 2 min for equilibration with the membrane. Centrifuge the column at 10,000 x g for 1 min. * Do not centrifuge above 10,000 x g, which may break the centrifuge tube. 8. Discard the filtrate, and load the remaining cleared lysate, if any, into the same Midi-Spin Column. Repeat centrifugation and discard the filtrate. 9. Add 10 ml Endotoxin Removal Wash Solution (isopropanol added) to the Midi-Spin column, wait for 2 min for equilibration with the membrane and centrifuge at 10,000 x g for 1 min. Discard the filtrate. 10. Add 10 ml Wash Solution to the column, and wait for 2 min for equilibration with the membrane. Centrifuge at 10,000 x g for 1 min and discard the filtrate. Repeat this step once more. 11. Reassemble the column to the tube and centrifuge at 10,000 x g for 5 min to dry the column. 12. For complete evaporation of ethanol, incubate the column at 45~60 C oven for 10 min. * Over-drying of the column (> 10 min) may result in poor elution of DNA. 13. To elute the DNA, place the Midi-Spin column into a sterile 50 ml centrifuge tube and add 1 ml preheated (65~70 C) Elution Solution or TE or H 2 O (ph 7.0~8.5) over the membrane. Wait for 3 min and centrifuge at 3
10,000 x g for 5 min to elute DNA. Repeat this step once more to increase DNA yield. 14. Collect the DNA solution into 2.0 ml or 3.0 ml microcentrifuge tube and store at -20 C. *Notes: To increase the DNA yield and concentration, user may elute an additional 1 ml (i.e. total 3 ml). 4
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. 5
f) Poor cell lysis 1) Excessive growth of bacteria. The culture 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 pre-heated Elution solution (60~70 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 to 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 6
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 3) Reduce the culture volume, if necessary. 7
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