Isolation of plasmids, using:
Our modified Qiagen plasmid mini kit protocol
Materials:
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Plasmid mini kit catalog number: 12125
Kubota 3500 centrifuge
BECKMAN COULTER Allegra X-22R centrifuge
Isopropanol
70-75% ethanol
Kit content
BUFFER
Buffer P1(resuspension buffer)
Buffer P2 (lysis buffer)
Buffer P3 (neutralization buffer)
Buffer QBT (equilibration buffer)
Buffer QC (wash buffer)
Buffer QF (elution buffer)
COMPOSITION
50 mM Tris-Cl, pH 8.0;
10 mM EDTA;
100µg/ml RNase A
200 mM NaOH, 1% SDS (w/v)
3.0 M potassium acetate, pH 5.5
750 mM NaCl;
50 mM MOPS, pH 7.0;
15% isopropanol (v/v);
0.15% Triton® X-100 (v/v)
1.0 M NaCl;
50 mM MOPS, pH 7.0;
15% isopropanol (v/v)
1.25 M NaCl;
50 mM Tris·Cl, pH 8.5;
15% isopropanol (v/v)
STORAGE
2-8˚C, after addition
of RNase A
15–25°C
15–25°C or 2–8°C
15–25°C
15–25°C
15–25°C
Things to do before starting:
 Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase A
(centrifuge briefly before use) per bottle of Buffer P1, to give a final concentration of
100µg/ml.
 Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary,
dissolve the SDS by warming to 37°C.
 Pre-chill Buffer P3 at 4°C.
Procedure:
1. Pick a single colony from a selective plate and grow the cells in 10 ml LB medium
and 10µl ampicillin. Incubate for 8-16 hours at 37˚C with vigorous shaking. Use the
50 ml tubes.
2. After incubating, weigh and find tubes that have the same weight for centrifuging. Use
the BECKMAN COULTER Allegra X-22R centrifuge, and centrifuge at 4500 rpm for
15-18 min at 4˚C.
3. Remove the liquid, leave the pellet in the tube (Collect the waste liquid in another tube
for risk waste container).
4. Resuspend the bacterial pellet in 600 µl of Buffer P1, vortex until everything is
dissolved.
5. Add 600 µl of Buffer P2. Mix by inverting the tube 2-3 times. Do not vortex, as this
will result in shearing of genomic DNA. Incubate in room temperature for 5 min. Do
not allow the lysis reaction to proceed for more than 5 min.
6. Add 600 µl of Buffer P3 (stored on ice). Mix immediately by inverting the tube 2-3
times. Incubate on ice for 5 minutes.
7. Transfer the sample from the 50 ml tube to an 2 ml eppendorftube. Use the Kubota
3500 centrifuge, and centrifuge at 15.000 rpm for 5 minutes at 4˚C.
8. While centrifuging, set up a QIAGEN-tip 20 column over a waste tray, and equilibrate
by applying 1 ml of Buffer QBT to the column. Allow the column to empty by gravity
flow.
9. When the centrifuge from step 7 has finished, transfer the liquid to the column
prepared in step 8. Allow the liquid to enter the resin by gravity flow.
10. Wash the QIAGEN-tip 20 with 2 x 2 ml of Buffer QC. Allow Buffer QC to move
through the QIAGEN-tip by gravity flow.
11. Place the QIAGEN-tip in an 1,5 µl eppendorftube, and elute DNA with 800 µl Buffer
QF. Collect the flow-through in the eppendorftube.
12. To the eppendorftube, add 540 µl isopropanol and mix well. Centrifuge at 15.000 rpm
for 30 minutes at 4˚C, in the Kubota 3500 centrifuge. Remember to place the tubes in
the same direction; the pellet can be difficult to see.
13. Take the samples carefully out of the centrifuge, remove the liquid. Add 1 ml 70-75 %
ethanol, centrifuge at 15.000 rpm for 5-10 minutes at 4˚C in the Kubota 3500
centrifuge.
14. After centrifuging, remove the ethanol. Leave the tubes with the lid open until the
ethanol has evaporated.
15. Dissolve the DNA in 20 µl dH2O.
16. Store the samples in the -20˚C freezer.