Oct 24, 2019
Miracle Prep for Plasmid Isolation
- 1University of Chicago
Protocol Citation: Blake Flood 2019. Miracle Prep for Plasmid Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.8p2hvqe
Manuscript citation:
Pronobis MI, Deuitch N, Peifer M (2016) The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields. PLoS ONE 11(8): e0160509. https://doi.org/10.1371/journal.pone.0160509
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
We use this protocol and it's working
Created: October 24, 2019
Last Modified: October 24, 2019
Protocol Integer ID: 29146
Keywords: DNA isolation, cloning, plasmid prep
Materials
MATERIALS
Sterile deionized H2O
LB Broth
Buffer P1QiagenCatalog #19051
Buffer P2QiagenCatalog #19052
Buffer N3QiagenCatalog #19064
96% EtOH
Econospin Mini Spin Column 250ctEpoch Life ScienceCatalog #1920-250
Buffer PBQiagenCatalog #19066
Buffer PEQiagenCatalog #19065
STEP MATERIALS
Buffer P1QiagenCatalog #19051
Buffer P2QiagenCatalog #19052
Buffer N3QiagenCatalog #19064
Buffer PBQiagenCatalog #19066
Protocol materials
96% EtOH
Econospin Mini Spin Column 250ctEpoch Life ScienceCatalog #1920-250
Buffer PBQiagenCatalog #19066
Buffer P1QiagenCatalog #19051
Sterile deionized H2O
LB Broth
Buffer P2QiagenCatalog #19052
Buffer P2QiagenCatalog #19052
Buffer PEQiagenCatalog #19065
Buffer N3QiagenCatalog #19064
Buffer PBQiagenCatalog #19066
Buffer P1QiagenCatalog #19051
Buffer N3QiagenCatalog #19064
Buffer P1QiagenCatalog #19051
Buffer P2QiagenCatalog #19052
Buffer N3QiagenCatalog #19064
Buffer PBQiagenCatalog #19066
Grow bacteria containing plasmid
Grow bacteria containing plasmid
Pick colony from plate. Inoculate 50 mL LB broth per vector.
Allow to grow overnight at 37 °C .
Isolate Plasmid
Isolate Plasmid
Spin culture 00:10:00 4000 x g 4 °C
Isolate Plasmid
Isolate Plasmid
Remove supt. Resuspend cells in 2 mL Buffer P1.
Buffer P1QiagenCatalog #19051
.
Isolate Plasmid
Isolate Plasmid
2 mL Buffer P2 (invert 3-4 times)
Buffer P2QiagenCatalog #19052
Incubate 00:03:00 @ Room temperature
2 mL Buffer N3 (invert 3-4 times)
Buffer N3QiagenCatalog #19064
Distribute lysate into 4 x 1.5mL tubes
Centrifuge 00:10:00 @ 13200 x g
Collect supt, add 1 volume 96% EtOH, mix
Load on 4 x “QIAprep 2.0 spin columns” or any generic silica membrane mini spin column (Econospin).
Spin 00:01:00 @17900 x g
Re-load and re-spin until everything is loaded.
500 µL Buffer PB to each column
Buffer PBQiagenCatalog #19066
Spin 00:01:00 @17900 x g
750 uL Buffer PE
Spin 00:01:00 @17900 x g
Spin again to dry 00:01:00 @17900 x g
Elute with 30-50 uL H2O
(Optional) Re-load H2O on columns and re-spin 00:01:00 @17900 x g to increase yield.