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Extract DNA (Maxiprep or Midiprep)
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17. Run on the agarose gel for as long as required to obtain maximum resolution. | 17. Run on the agarose gel for as long as required to obtain maximum resolution. | ||
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==Maxiprep Protocol (QIAGEN kit)== | ==Maxiprep Protocol (QIAGEN kit)== | ||
Revision as of 18:31, 28 October 2008
Midiprep Protocol (QIAGEN kit)
1. Transfer the overnight culture of plasmid cells into 2ml microcentrifuge collection tubes (1 per try). Pellet for 3 min at >8000 rpm in a conventional table-top microcentrifuge at room temperature. Decant all the liquid.
2. Add 250ul of ice cold resuspension Buffer P1 (make sure RNase is already added and mixed. Buffer P1 is stored at 4C)
3. Add 250 uL of Buffer P2 and mix thoroughly by inverting the tube 4-6 times for exactly 5 min: no more, no less. Mix gently. DO NOT vortex. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after the addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If there are still clumps, keep mixing until the homogeneous suspension is achieved.
4. Add 350 uL of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution should become cloudy. If LyseBlue has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless.
5. Centrifuge for 10 min at 13,000rpm (17900 g) in a table-top microcentrifuge. A compact white pellet will form.
6. Setup the QIAprep spin column
7. Transfer the supernatants from step 5 to the QIAprep spin column by decanting or pipetting.
8. Centrifuge for 1 min at 13,000 rpm. Discard the flow through.
9. Wash the QIAprep spin column by adding 0.5 mL Buffer PB. Centrifuge for 1 min at 13,000 rpm. Discard flow-through.
10. Wash QIAprep spin column by adding 0.75 mL Buffer PE. Centrifuge for 1 min at 13,000rpm
11. Transfer the filters to a new clean and autoclaved 2ml eppendorf tube.
12. Label the tubes.
13. Add 50 µl of EB per column and wait 3 min.
14. Spin for 5 mins.
15. Measure the concentration of DNA.
16. Digest the tries which seem to have the correct supercoil size with an appropriate restriction enzyme to verify plasmid construction. Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme. You need atleast 100ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 1 µg as they will be divided as 900ng of the 4.5kb band and 100ng of the 500 bp band.
17. Run on the agarose gel for as long as required to obtain maximum resolution.
Maxiprep Protocol (QIAGEN kit)
All centrifugation steps are done at 4000 g, 4C unless otherwise specified.
1. Transfer 400ml of the overnight culture of plasmid cells into two clean, autoclaved 200ml centrifuge bottles (1 per plasmid). Pellet for 15 min at 6000 g, 4C. Decant all the liquid. Make sure not to mix up the plasmids.
2. Add 10ml of Resuspension Buffer P1 to the cell pellet and vortex the bottle. No cell clumps should be observed.
3. Add 10ml of Lysis solution Buffer P2. Do not vortex. Mix gently by swirling the bottle. If LyseBlue was added to Buffer P1, the cell suspension will turn blue after the addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
4. Add 10ml of chilled Neutralization solution Buffer P3. Do not vortex. Mix gently by swirling the bottle. A white precipitate will appear. If LyseBlue was used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless.
5. Centrifuge the cells for 30 mins at ≥20,000 g, 4C. Remove supernatant containing plasmid DNA promptly. Centrifugation should be performed in non-glass tubes. After centrifugation the supernatant should be clear.
6. Centrifuge the supernatant again for 15 mins at ≥20,000 g, 4C. Remove supernatant containing plasmid DNA promptly. Second centrifugation should be carried out to avoid applying suspended or particulate material to the QIAGEN-tip. Suspended material can clog the QIAGEN-tip and reduce or eliminate gravity flow.
7. Add 10ml of the matrix per bottle. Swirl and mix for 30 sec. Centrifuge for 5 min. The matrix binds the DNA and settles at the bottom.
8. Throw out the supernatant. Resuspend the matrix in 25 ml of wash buffer. Centrifuge for 5 min. Make sure that the wash buffer bottle is marked ‘ethanol added’. If not, add 100% ethanol to fill the wash buffer bottle.
9. Assemble the filters. Place each filter provided in the kit in a 50ml centrifuge tube (blue cap, also provided in the kit). Label the tubes.
10. Throw out the supernatant. Resuspend the wash buffer in 15 ml of the wash buffer and mix thoroughly. Transfer the solution to the corresponding filter/tube from the above step.
11. Centrifuge for 5 min.
12. Remove the filter and throw out the solution. Place the filter back in the same tube. Add 10 ml of wash buffer and centrifuge for 5 min.
13. Remove the filter and place it in a new 50 ml centrifuge tube (provided in the kit). Label the tube and add 4 ml of EB (elution buffer) to the filter. Let it stand for 3 min at RT.
14. Centrifuge for 5 min.
15. Discard the filters from each tube. Add 222 µl of 5M NaCl to each tube. Mix well by swirling gently. Add 8ml of ice cold 100% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min.
16. You will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet.
17. Add 10 ml of 70% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min.
18. Once again, you will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet.
19. Air dry the pellet (leave it on your bench for 10-15 min.) Don’t leave the pellet for a longer duration as it might be difficult to resuspend.
20. Resuspend the pellet in 1.5 ml of EB.
21. Measure the concentration and aliquot into labeled 2ml Eppendorf tubes.
22. Store the DNA at -20C.
