HighPure Maxi Plasmid Kit

Transcription

1 HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields

2 PP HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer P1 Buffer P2 Buffer P4 Buffer TB TIANRed DP preps 500 μl 100 ml 100 ml 100 ml 30 ml 500 μl Filtration Columns CS1 10 Handbook 1 HighPure Maxi Plasmid Kit can be stored dry at room temperature (15-25 C) for up to 12 months without showing any reduction in performance and quality. For longer storage, it can be stored at 2-8 C. If any precipitate forms in the buffers after storage at 2-8 C, it should be dissolved by warming the buffers to 37 C before use. RNaseA (100 mg/ml) can be stored for one year at room temperature (15-25 C). After adding RNaseA, Buffer P1 is stable for 6 months at 2-8 C. Introduction HighPure Maxi Plasmid Kit adopts specially designed buffer system that is used in conjunction with isopropanol precipitation to achieve fast and efficient purification of high pure plasmid DNA. Plasmid DNA prepared by HighPure Maxi Plasmid Kit is suitable for a variety of routine applications including restriction enzyme HighPure Maxi Plasmid Kit Handbook 1

3 digestion, sequencing, library screening, ligation and transformation, in vitro translation, and transfection of cell lines. Recommended bacterial culture volume: ug plasmid with 100 ml bacterial culture for high-copy vectors; and ug plasmid with 200 ml bacterial culture for low- copy vectors. Using TIANRed reagent TIANRed reagent is a color indicator which provides visual identification of optimum buffer mixing. This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. Researchers can determine whether to use TIANRed according to their experience and experiment purpose. TIANRed has no influence on following reactions, such as PCR, enzyme digestion and sequencing. If the purified plasmid is to be used in transfection experiment, however, usage of TIANRed is not recommended. TIANRed should be added to the Buffer P1 before use at a ratio of 1:200. Mix thoroughly by inverting, and the solution will present transparent red. The plasmid preparation procedure is performed as usual. After add Buffer P1 to harvested bacterial cells, the suspension will become cloudy. After addition of Buffer P2 to Buffer P1 the color of the suspension changes to purple. Mixing should result in a homogeneously transparent purple suspension, showing the lysis is complete. Upon addition of Buffer P4, TIANRed turns transparent yellow. The presence of a homogeneous solution with no traces of purple indicates that SDS from the lysis buffer (Buffer P2) has been effectively precipitated. Important notes 1. Prevent to contact Buffer P2 and P4 directly. Close the bottle containing Buffer P2 and P4 immediately after use to avoid acidification of Buffer P2 and P4 from CO 2 in the air. 2. Draw out the plunger from the filter slowly to avoid membrane HighPure Maxi Plasmid Kit Handbook 2

4 loose. 3. The amount of extracted plasmid is related to cells concentration and plasmid copy. If working with low copy vectors or large plasmid (>10 kb), it may be beneficial to increase culture volume and to increase Buffer P1, P2, and P4 in proportion. Warm the Buffer TB to C before use. Prolong adsorption and elution time properly to increase extraction efficiency. Things to do before starting 1. Add the provided RNase A solution (500 μl) to Buffer P1, mix, and store at 2-8 C. 2. Check Buffer P2 and P4 for SDS precipitation due to low storage tempertures. Redissolve the precipitate by warming to 37 C. Do not shake Buffer P2 vigorously. 3. Prepare 60 ml 5 M NaCl solution. 4. Optional: Add the provided TIANRed reagent to Buffer P1 before use at a ratio of 1:200. If the purified plasmid is to be used in transfection experiment, however, usage of TIANRed is not recommended. Protocol 1. Harvest 100 ml bacterial culture cells by centrifuging at 10,000 rpm (~11,500 g) for 3 min at room temperature (15-25 C), and then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained. 2. Resuspend pelleted bacterial cells in 10 ml Buffer P1. Note: Ensure that RNase A has been added to Buffer P1. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. If TIANRed reagent has been added to Buffer P1, mix HighPure Maxi Plasmid Kit Handbook 3

5 thoroughly before use. The suspension become cloudy red due to the presence of bacteria cells. 3. Add 10 ml Buffer P2 and mix thoroughly by inverting the tube 6-8 times, and incubating at room temperature for 5 min. Note: Mix by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If TIANRed has been added to Buffer P1 the cell suspension will turn purple after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized red regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved. 4. Add 10 ml Buffer P4, and mix immediately and thoroughly by vigorously inverting 6-8 times, until the whole solution become cloudy. Incubate at room temperature for 10 min. A white fluffy material forms. Note: To avoid localized precipitation, mix the lysate thoroughly and immediately after addition of Buffer P4. Mix by inverting the tube. Do not vortex. If TIANRed has been used, the suspension should be mixed until all trace of perple has gone and the suspension become transparent yellow. A homogeneous yellow suspension indicates that the SDS has been effectively precipitated. 5. Centrifuge for 10 min at 10,000 rpm (~11,500 g). The white material should be precipitated to the bottom of the centrifuge tube and form a white pellet. Note: The centrifugation time can be prolonged appropriately to precipitate the white material. If use more than 100 ml HighPure Maxi Plasmid Kit Handbook 4

6 bacterial culture, prolong centrifugal time to min. 6. Transfer the supernatant into a Filtration Column CS1. Gently insert the plunger into the Filtration Column CS1 and filter the cell lysate into a new 50 ml tube (not supplied in the kit). Note: If the white material fails to be precipitated to the bottom in step 5, it means the lysate had not been mixed thoroughly in step 5. In this scenario, avoid transferring large clump into the Filtration Column CS1, which will clog the filtration membrane. Small fragments have on influence on Filtration Column CS1. 7. Add 0.35 volume isopropanol to the cleared lysate, and then add 5M NaCl of half amount of the isopropanol. Sealed the received tube, and mix thoroughly by inverting the tube. Note: it is a normal phenomenon if no visible precipitate appears in this step. 8. Centrifuge at 10,000 rpm (~11,500 g) for 30 min at 4 C. Decant all the supernatant. 9. Wash the a pellet with 6 ml 70% ethanol. Centrifuge at 10,000 rpm (~11,500 g) for 10 min at 4 C. Decant all the supernatant without disturbing the pellet. 10. Repeat the step Air dry the plasmid DNA pellet for minutes. And redissolve the DNA in ml Buffer TB. Note: Overdrying the pellet will make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline conditions. Buffer TB or distilled water (ph ) is recommended to be used to redissolve the plasmid DNA. For long-term storage of DNA, dissolving in Buffer TB and storing at 20 C can avoid the possible acid hydrolysis of DNA in water. HighPure Maxi Plasmid Kit Handbook 5

7 Ordering Information Transfection Reagent Product Size Cat.no. Lipofect Transfection Reagent 0.5 ml 5 ml RM RM DNAfectin Transfection Reagent 0.75 ml ml RM RM Plasmid DNA Purification Product Size Cat.no. TIANprep Mini Plasmid Kit 50 preps 200 preps DP DP EndoFree Maxi Plasmid Kit 10 preps DP117 HighPure Maxi Plasmid Kit Handbook 6