Dec 01, 2016
Column-free plasmid miniprep
- Vladimir Vigdorovich1
- 1Center for Infectious Disease Research
Protocol Citation: Vladimir Vigdorovich 2016. Column-free plasmid miniprep. protocols.io https://dx.doi.org/10.17504/protocols.io.d7e9jd
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 13, 2015
Last Modified: February 03, 2018
Protocol Integer ID: 1990
Abstract
This is a protocol for plasmid minipreps that does not involve any columns and uses commonly accessible reagents.
While it doesn't result in highly pure DNA, it can provide a sufficient quantity of plasmid for sequencing, restriction analysis, preparative digests.
Beware of the large amount of RNA that co-purifies with plasmid DNA (it acts as an efficient carrier during the nucleic acid precipitation step). This contamination will make it impossible to determine DNA concentration from standard 280-nm absorbance readings.
Credits: I first learned this protocol from the old-style protocols for bacmid purification using the Bak-to-Bac system (Invitrogen).
In that system, the large (>50 kb) bacmid molecules were deemed too fragile for the Qiagen spin columns.
Guidelines
Buffer P1
* 50 mM Tris-HCl pH 8.0
* 10 mM EDTA
* 100 ug/ml RNaseA
The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101).
Note: LyseBlue reagent (1000X = 43 mg/ml thymolphthalein in ethanol)
Buffer P2
* 200 mM NaOH
* 1% SDS
Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits; N3 contains the equilibration buffer, while P3 does not)
* 3.0 M potassium acetate pH 5.5
Materials
MATERIALS
Buffer P1
Buffer P2
Buffer P3Catalog #19053
Isopropanol
1.5 mL Eppendorf tubes
nuclease-free water
Culture growth
Culture growth
Grow an overnight 3-mL E. coli culture in LB with appropriate antibiotics. Preferably, in round-bottom snap-cap culture tubes suitable for centrifugation (in the next step).
3 µL
Culture harvesting
Culture harvesting
Spin down at 4,000 rpm in a table-top centrifuge for ~15 min. Discard supernatant and vortex pellets to resuspend in residual culture medium.
00:15:00
Resuspension
Resuspension
Add the roughly resuspended cells to 200 µL Buffer P1. Mix by pipetting or vortexing.
200 µL
Lysis
Lysis
Add 200 µL Buffer P2.
Mix gently by inverting the tube several times.
200 µL
Neutralization
Neutralization
Add 350 µL Buffer P3.
Mix vigorously by inverting/shaking the tube several times. Do not vortex.
Be sure to thoroughly mix the viscous solution from previous step and allow the flocculant SDS precipitate to form.
350 µL
Lysate clarification
Lysate clarification
Centrifuge for 5 min in a microcentrifuge (>10,000 rpm) to remove SDS and cell debris.
00:05:00
Nucleic acid precipitation
Nucleic acid precipitation
Transfer the clarified lysate (~750 µL) to a clean tube containing 750 µL pure isopropanol.
Mix by inversion.
750 µL
Nucleic acid isolation
Nucleic acid isolation
Centrifuge for 5 min in a microcentrifuge (>10,000 rpm) to pellet nucleic acids.
00:05:00
Nucleic acid pellet cleanup
Nucleic acid pellet cleanup
Wash the nucleic acid pellet with 500 µL of 70% ethanol.
Gently dislodge the nucleic acid pellet from the side of the tube by shaking.
500 µL
Nucleic acid pellet cleanup: salt removal
Nucleic acid pellet cleanup: salt removal
Centrifuge for 5 min in a microcentrifuge (>10,000 rpm) to pellet nucleic acids.
00:05:00
Nucleic acid pellet cleanup: ethanol removal
Nucleic acid pellet cleanup: ethanol removal
Gently pipette off the ethanol and air-dry the nucleic acid pellet.
If you have a SpeedVac centrifuge, use it for ~2 min.
Don't overdry the pellets, as that will cause problems with resuspension.
Nucleic acid pellet resuspension
Nucleic acid pellet resuspension
Dissolve the pellet in 50 µL nuclease-free water.