Plasmid DNA Extraction (Minipreps)
[Note: the standard “miniprep” protocol to extract plasmids out of bacteria may vary depending
on which proprietary kit is used, but is generally the same procedure.]
Kit used: ThermoFisher “GeneJET Plasmid Miniprep Kit” (Catalog No. FERK0503, FisherSci)
1. Grow E. coli colony (single clonal isolate required!) in 2xLB+Amp or Kan culture overnight for
12-16 hours at 30°C or 37°C until saturated. [Note: important only a single colony is taken for
inoculating the culture; if colonies are too close together, or in a “lawn,” streak on selection
plates a second time to obtain separated, single isolates. Use a sterile toothpick/stick to
inoculate the culture. Can leave the stick in the culture overnight.]
2. Discard the stick/pick. Transfer 1.5 mL of culture to Eppendorf tube and spin at max for 1
minute.
3. Discard media safely (keep pellet).
4. Add a second 1.5 mL of culture to the SAME tube. [Note: Take extreme care to only transfer
a second aliquot of the same culture to the same tube. Do NOT mix this up! Take care to label
everything carefully. If multiple isolates from the same plasmid are being tested, then label each
uniquely (e.g. 100-1, 100-2, 100-3, etc.) on both the overnight culture tube AND on the
Eppendorf tubes.]
5. Spin 1 minute at max speed.
6. Discard media safely (keep pellet).
7. Can use pipette tip to remove the final amount of media from tube, leaving only pellet.
8. Add 250 uL of “Resuspension Buffer” (stored at 4°C fridge; ensure that this buffer already has
the RNAaseA added; see miniprep kit instructions) and resuspend by pipetting up and down
until pellet is gone and solution is cloudy.
9. Vortex for 10-15 seconds to ensure complete mixing. Do not leave any chunks or portions of
pellet left.
10. Ensure that the “Lysis Buffer” is clear and mixed. [Note: Often, at lower temperatures
(transport from company, or stored in AC-environment, or in winter, etc.) solids may precipitate
out of solution in this buffer. To fix, place the bottle at 37°C for 10-15 minutes to warm, and mix
the bottle by swirling until ALL precipitates are gone and back into solution. Use of the lysis
buffer if anything is out of solution causes the procedure to fail.]
11. Add 250 uL of Lysis Buffer to each tube with a fresh tip every time.
12. Mix the tubes by inversion 6-10 times gently.
13. Incubate at room temp for 4.5-5 minutes.
14. Add 350 uL of “Neutralization Buffer” to each tube. [Note: After lysis buffer has been added,
the solution should turn clear and will become very viscous. Be sure when opening the tube, no
material gets out. Also, a bubble may form on the lid of the tube. After 4 minutes of lysis, open
the tube, and within a minute the bubbles should have popped allowing for the next step. Do
NOT add neutralization buffer to the tube if a bubble of material blocks the opening.]
15. Mix by inversion 10-20 times. White precipitate forms.
16. Immediately spin at max for 5 minutes.
17. Carefully pour the liquid (no precipitate!) into the provided Miniprep Spin Columns.
18. Spin at max for 1 minute.
19. Discard the flow-through.
20. Add 500 uL of “Wash Buffer.” [Note: once the kit has been opened, add 170 uL of 96%100% ethanol to the wash buffer and mark this on the top of the bottle].
21. Spin at max for 1 minute.
22. Discard the flow-through.
23. Add 500 uL of Wash Buffer a second time.
24. Spin at max for 1 minute.
25. Discard the flow-through.
26. Spin the EMPTY column for 1 minute. [Note: this “empty” spin is extremely important to all
miniprep protocols and is used to remove all traces of ethanol from the prep. DO NOT forget the
empty spin!]
27. Remove the column from the bottom portion (toss away) and place the column into a fresh,
labeled Eppendorf tube.
28. Add 65 uL of “Elution Buffer” directly to the filter (without touching it).
29. Let sit at room temp on bench for 1 minute.
30. Spin at max for 2 minutes.
31. Discard the column and keep the Eppendorf tube with your purified plasmid DNA in it.
[Note: at this point, store the plasmid miniprep at either 4°C in your fridge, or at -20°C for longterm storage. Can use the NanoDrop Lite to determine the concentration of DNA (100-300
ng/uL is typical). Purified miniprep DNA is ready for use in digestions, PCR, transformation into
yeast, transformation into bacteria, or other uses.]
[Additional Notes: Pink colonies: Constructs that contain mCherry in them sometimes cause
the TOP10 bacterial colonies to turn pink/red in color! So, if you see this, don’t freak out; the
construct has mCherry expressed (somehow) and is likely correct.]
[Additional Notes: Poor DNA yields: For some (rare) genes, such as BNI5, plasmids that
contain the full-length gene are TOXIC—the presence of the BNI5 gene in the plasmid construct
(either yeast-based or bacterial based) causes small colonies, slow growth of colonies, a lighter
shade (rather than dark opaque color) of colony, and very very low yields using ANY miniprep
kit. This seems to be rare, but can definitely occur for some genes.]
[Additional Notes: Yeast plasmid preps: For isolating plasmids from yeast, please see “Yeast
Plasmid Prep” protocol which is a slight variant on this miniprep procedure, yet uses the same
kit and principles.]
[Additional Notes: Opening a new kit: The kits require a few simple preparation steps including
(i) Adding the RNAaseA solution to the Resuspension Buffer and storing at 4°C, (ii) Adding
ethanol to the wash buffer, and (iii) ensuring that the Lysis Buffer is mixed and clear with no
precipitates (see Notes above at Step 10).]