"
Extract DNA (Miniprep)
From 2008.igem.org
PRINCETON IGEM 2008
| Home | Project Overview | Project Details | Experiments | Results | Notebook |
|---|
| Parts Submitted to the Registry | Modeling | The Team | Gallery |
|---|
Miniprep Protocol (QIAGEN Miniprep kit)
Minipreps are used to extract the DNA from the grown bacteria.
Miniprep Growth Protocol
1. Calculate the approx. S/N ratio of the transformation.
2. Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise.
3. Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available.
4. Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.)
5. Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube.
6. Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try.
7. Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.
Miniprep Extract DNA Protocol
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.
