Plasmid Maxiprep Plus Purification Kit

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1 Plasmid Maxiprep Plus Purification Kit Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only 1

2 Description: The Plasmid Maxiprep Plus Purification Kit provides simple, rapid protocol and materials to purify plasmid DNA from 200~800 ml liquid culture of via the spin column format. The yield of DNA, from 0.5~1.5 mg, is quantitative and reproducible. The obtained DNA, which is free of RNA, endotoxin and other expected contaminants, can be used for most subsequent applications, including polymerase chain reaction, restriction digestion, cloning, DNA sequencing, hybridization, in vitro transcription and transfection. Component of the Kit: DP01MX-P10 DP01MX-P20 1. Solution I 110 ml 220 ml 2. Solution II 110 ml 220 ml 3. Solution III 110 ml 220 ml 4. Endotoxin Removal 72 ml (add 48 ml 144 m l (add 96 ml Wash Solution isopropanol before use) isopropanol before use) 5. Wash Solution 48 ml (add 192 ml 96 ml (add 384 ml Ethanol before use) Ethanol before use) 6. Elution Solution 40 ml 80 ml 7. Maxi-Spin Column & Collection Tube 10 sets 10 sets x ml Collection Tube 10 pcs 10 pcs x 2 9. RNase A Powder* 40 mg 80 mg 10. Filter Net 10 pcs 10 pcs x 2 * Store RNase A Powder at -20 and all other kit components at room temperature (25~28 C). * If precipitates have formed in solution II, warm the buffer at 35 in a water bath until fully dissolved. Before starting this procedure: Dissolve RNase A Powder in Solution I: Add 1 ml Solution I to each tube of RNase A Powder, vortex until completely dissolve, transfer all the solution to the Solution I and store at 4. Add isopropanol [48 ml for DP01MX-P10 (or) 96 ml for DP01MX-P20] to 2

3 Endotoxin Removal Wash Solution and mix well. Store at RT. Add ethanol [192 ml for DP01MX-P10 (or) 384 ml for DP01MX-P20] to Wash Solution and mix well. Store at RT. Turn on water bath at 60~65. General Procedure: 1. Pellet down cells from 200~500 ml liquid culture by centrifugation at 12,000~14,000 x g for 5 min. Pour off the supernatant. * For low copy plasmid, the culture volume may be increased to 800 ml. 2. Completely resuspend the cell pellet in 10 ml Solution I by pipetting. Incubate at RT for 10 min for uniform and complete suspension of cells especially for high density (> 3 x 10 9 cells/ml) culture or large volume > 250 ml culture. 3. Add 10 ml 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 10 ml Solution III and mix by inverting the tube 6~8 times. Incubate on ice for 10 min. 5. Centrifuge the lysate at 12,000~14,000 x g for 20 min at 4. A compact white pellet will be formed along the side or at the bottom of the tube. 6. To remove the floating pellet, hold a Filter Net by hand above a sterile 50 ml centrifuge tube (not provided) and gently pour the lysate through the net. See the figure below. 7. Open the cap of a Maxi-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 3

4 min for equilibration with the membrane. Centrifuge the column at 12,000x g for 1 min. * Do not centrifuge above 12,000x g, which may break the centrifuge tube. 8. Discard the filtrate, and add the remaining cleared lysate, if any, into the same Maxi-Spin Column. Repeat centrifugation and discard the filtrate. 9. Add 10 ml Endotoxin Removal Wash Solution (isopropanol added) to the Maxi-Spin column, wait for 2 min for equilibration with the membrane and centrifuge at 12,000x 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 12,000x g for 1 min and discard the filtrate. Repeat this step once more. 11. Reassemble the column to the tube and centrifuge at 12,000x g for 10 min to dry the column. 12. For complete evaporation of ethanol, incubate the columnat 60~65 C oven for 15 min. * Over-drying of the column (> 15 min) may result in poor elution of DNA. *Please only incubate the column (without 50 ml centrifuge tube) in the oven, or the ethanol could not be evaporated completely. 13. To elute the DNA, place the Maxi-Spin column into a sterile 50 ml centrifuge tube and add 2-3 ml preheated (60~65 C) Elution Solution or TE or H 2 O (ph 7.0~8.5) to the membrane. Let stand for 3 min and centrifuge at 12,000 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). This may increase the concentration and yield of plasmid DNA. 4

5 Appendix: DNA Concentration by ethanol precipitation 1. Prepare suitable size of centrifuge tube which can be used at >15,000 x g. 2. Save about 10 to 20 μl DNA eluate as control to check the DNA loss after precipitation. 3. Add 1/10 volume of 3 M sodium acetate (ph 5.2) or 1/3 volume of 10 M ammonia acetate to plasmid DNA, mix well. 4. Add 2 volumes of ice-cold ethanol to the solution, mix well and incubate at -20 > 60 min or at -80 for 20 min. 5. Centrifuge at > 15,000 x g for 30 min at 4 C. 6. Carefully remove the supernatant without disturbing the pellet (some of DNA pellet may stick along the wall of tube). 7. Wash the pellet with 10 ml ice-cold 70% ethanol, and centrifuge at > 15,000 x g for 10 min at 4 C. 8. Carefully remove as much supernatant as possible and air dry the DNA pellet. 9. Re-dissolve the DNA pellet with water or TE buffer, rinse along the wall of the tube to some DNA pellet on the wall of the tube. 5

6 Troubleshooting Guide Comments and Suggestion Low or no yield a) Plasmid did not Inoculate the liquid medium containing propagate 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) Spin-column is clogged Do not use more sample volume than recommended to prevent clogging of spin-column d) Not enough bacterial cells Ensure optimum growth of culture (OD 600 > 1) by incubating the culture overnight at recommended temperature with vigorous shaking 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 medium should contain recommended antibiotic. 2) Poor cell suspension. Ensure complete resuspension of bacterial pellet by votexing 6

7 g) Incomplete DNA Elution or pipetting before adding Solution II. 1) If plasmid is larger than 7 Kb, use preheated Elution solution (60~65 ) in the Step 13. 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 DNA shearing. 2) Lysis in step 3 must not exceed 5 minutes. c) Solution III added incorrectly RNA contamination RNase A digestion insufficient Upon addition of Solution III, mix immediately, but gently. 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 7

8 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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