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Extract DNA (Miniprep)
From 2008.igem.org
(New page: ==Miniprep Protocol (QIAGEN Miniprep kit)== All centrifugation steps are done at 13000 rpm, after step 2, in a table-top microcentrifuge 1. Transfer the overnight culture of plasmid cel...) |
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==Miniprep Protocol (QIAGEN Miniprep kit)== | ==Miniprep Protocol (QIAGEN Miniprep kit)== | ||
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All centrifugation steps are done at 13000 rpm, after step 2, in a table-top microcentrifuge | All centrifugation steps are done at 13000 rpm, after step 2, in a table-top microcentrifuge | ||
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Revision as of 01:28, 29 October 2008
Miniprep Protocol (QIAGEN Miniprep kit)
Minipreps are used to extract the DNA from the grown bacteria.
All centrifugation steps are done at 13000 rpm, after step 2, in a table-top microcentrifuge
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.
