Plasmid separation (Miniprep)
Before You
Begin
-
If the Cell Lysis Buffer
appears cloudy or has precipitates, warm it at 30–37°C for 30 minutes and mix
gently by inversion.
-
Always work at room temperature
unless noted.
-
Pelleting cells before lysis
improves DNA purity.
Steps
1. pelleting
-
Centrifuge the 5 mL culture at
max speed for 10 minutes.
-
Discard the supernatant
carefully.
-
Re-suspend in 600 µL of TE
Buffer.
-
Transfer it into 1.5 mL
microcentrifuge tube.
2. Cell Lysis
-
Add 100 µL of Cell Lysis
Buffer.
-
Mix by inverting the tube 6
times (do not vortex).
-
The solution should turn clear
blue (indicating complete lysis).
-
Proceed to the next step within
2 minutes. Prolonged lysis can damage plasmid DNA.
3.
Neutralization
-
Add 350 µL of cold
Neutralization Solution (4°C).
-
Mix thoroughly by inverting the
tube until the solution turns yellow and a precipitate form.
-
Invert 3 more times to ensure
complete neutralization.
4. Clarify
Lysate
-
Centrifuge at maximum speed
(~13,000–14,000 rpm) for 3 minutes.
-
Carefully transfer the clear
supernatant (~900 µL) to a Minicolumn,
-
Place the minicolumn in a
collection tube
-
Centrifuge at maximum speed for
15 seconds.
-
Discard the flowthrough and
return the minicolumn to the same collection tube.
6. Wash Steps
-
Add 200 µL of Endotoxin Removal
Wash.
-
Centrifuge at max speed for 15
seconds.
-
Add 400 µL of Column Wash
Solution.
-
Centrifuge at max speed for 30
seconds.
7. Elute DNA
-
Transfer the minicolumn to a clean
1.5 mL microcentrifuge tube.
-
Add 30 µL of Elution Buffer or
Nuclease free water directly to the center of the column matrix.
-
Let it sit for 1 minute at room
temperature.
-
For large plasmids (>10 kb),
Warm elution buffer to 50°C and let sit for 5–10 minutes before centrifuging
8. Final
Elution
-
Centrifuge at maximum speed for
15 seconds.
-
Store your eluted plasmid DNA
at –30°C to –10°C.
5. Plasmid separation (Midi prep)
Materials
Required
-
NucleoBond® Xtra Midi Kit
(columns, filters, buffers RES, LYS, NEU, EQU, WASH, ELU)
-
RNase A (added to Buffer RES)
-
Isopropanol, 70% ethanol
-
TE buffer or sterile
nuclease-free water
-
15 mL or 50 mL centrifuge tubes
-
Tabletop centrifuge (≥ 4,500 ×
g)
-
Shaking incubator (37 °C)
-
Spectrophotometer (for DNA
quantification)
Steps
1. Bacterial
Cell Culture & Harvest
-
Prepare the starter culture of
LB medium with a single colony picked from a freshly streaked LB agar plate.
-
Inoculate overnight by diluting
the starter culture into 250ml LB broth at 37°C with shaking (200–300 rpm).
-
Centrifuge at 4,500–6,000 × g
for 10 min at 4°C.
-
Discard supernatant completely.
2. Cell
Resuspension
-
Resuspend pellet in 8 mL resuspension
buffer (with RNase A).
-
Mix thoroughly until no clumps
remain.
3. Cell Lysis
-
Add 8 mL Buffer LYS.
-
Gently invert the tube 5–6
times to mix (do not vortex).
-
Incubate at room temperature
for 5 minutes (no longer).
! Prolonged exposure/vortexing =
denaturation of DNA
-
If Lysis Buffer shows SDS
precipitates, warm it to 30–40°C until completely clear.
4.
Neutralization
-
Add 8 mL Buffer NEU.
-
Gently invert until the
solution turns colorless and precipitate forms.
-
Mix thoroughly but gently.
5. Column
Preparation
-
Equilibrate Nucleo Bond® Xtra
Column with 12 mL Buffer EQU.
-
Allow buffer to flow through by
gravity.
6. Lysate
Clarification and Loading
-
Gently invert lysate tube 3×
before loading to resuspend precipitate.
-
Apply the entire lysate to the
column with the column filter in place.
-
Let it flow through by gravity.
7. Wash Steps
-
Wash with 5 mL Buffer EQU (onto
filter rim).
-
Remove column filter and
discard.
-
Wash column with 8 mL Buffer
WASH.
8. Elution
-
Elute DNA using 5 mL elution buffer
into a clean centrifuge tube.
9. DNA
Precipitation
-
Add 3.5 mL isopropanol to
eluate.
-
Mix well and incubate at room
temperature for 2–5 minutes.
-
Centrifuge at 15,000 × g for 30
minutes at 4°C.
-
Discard supernatant carefully.
10. DNA Wash
and Drying
-
Wash pellet with 2 mL 70%
ethanol.
-
Centrifuge at 15,000 × g for 5
minutes.
-
Carefully remove ethanol.
-
Air-dry pellet at room temperature
for 10–15 minutes (avoid over-drying).
11. DNA
Reconstitution
-
Dissolve DNA in 200–800 µL TE
buffer or nuclease-free water.
-
Let sit at room temp or shake
gently for 15–30 minutes.
-
Measure DNA concentration using
Nanodrop.
-
Store DNA at –20°C.