Aug 11, 2022

Public workspaceMiniprep (NEB Monarch)

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
Brian Teague
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Protocol Citation: Brian Teague 2022. Miniprep (NEB Monarch). protocols.io https://protocols.io/view/miniprep-neb-monarch-ce5btg2n
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: August 10, 2022
Last Modified: August 11, 2022
Protocol Integer ID: 68483
Abstract
We transformed E. coli bacteria with our plasmid in order to make more of it -- to use the bacteria as highly accurate DNA copiers. Now that we've grown a bunch of E. coli, we need to get the plasmid DNA back out. That's the point of a miniprep.



Image Attribution
Lilly_M, CC BY-SA 3.0 , via Wikimedia Commons
Guidelines
It is easy to get "into a rhythm" with this protocol and miss or mis-read important steps. Follow the directions very carefully -- there are lots of buffers and centrifugation stepsS
Materials
Equipment
  • Vortexer
  • Microcentrifuge
  • Nanodrop (or equivalent instrument for measuring DNA quantity and purity)

Materials and Reagents
  • Microcentrifuge tubes
  • A 5 ml culture of E. coli A 5 ml culture of E. coli containing your plasmid (one per miniprep)
  • NEB spin column and collection tube (one per miniprep)
  • Resuspension buffer (red)
  • Lysis buffer (blue)
  • Neutralization buffer (yellow)
  • Wash buffer 1 (clear)
  • Wash buffer 2 (clear)
  • Elution buffer (clear)

(The spin columns and buffers come from the ReagentMonarch Plasmid Miniprep KitNEBCatalog #T1010 )

  • Biological waste container (50 ml conical)
  • Chemical waste container (50 ml conical)
Safety warnings
The chemicals in a miniprep are moderately hazardous, especially if you were to get them in your eyes. Wear appropriate PPE, including a lab coat, gloves and safety glasses.

Additionally, this protocol generates BOTH biological AND chemical waste. Follow your instructor's guidelines for disposing of these two waste streams.
Harvest the E.coli culture
Harvest the E.coli culture
1m
1m
Transfer Amount1.5 mL of bacterial culture to a microcentrifuge tube. Centrifuge Centrifigation16000 x g, 00:00:30

30s
Remove the supernatant using a micropipettor and discard in the biological waste container. Try to get as much as you can without disturbing the pellet.
Transfer another 1.5 ml of bacterial culture to the same microcentrifuge tube. CentrifugeCentrifigation16000 x g, 00:00:30

30s
Remove the supernatant using a micropipettor and discard in the biological waste container. Try to get as much as you can without disturbing the pellet.
Lyse the E.coli cells
Lyse the E.coli cells
8m
8m
Add Amount200 µL of resuspension buffer. Vortex to resuspend the pellet. Make sure it is completely resuspended -- there should be no visible clumps.

Add Amount200 µL lysis buffer. Invert the tube 4-6 times to mix and incubate at TemperatureRoom temperature for Duration00:01:00 . Do not vortex.

1m
Add Amount400 µL of neutralization buffer and gently invert the tube until neutralized. Sample is neutralized when the color is uniformly yellow. Do not vortex.

Incubate at TemperatureRoom temperature for Duration00:02:00

2m
Centrifuge lysate Centrifigation16000 x g, 00:05:00

5m
Bind and Wash
Bind and Wash
4m
4m
Carefully transfer the supernatant (the liquid at the top, not the pellet at the bottom) to the spin column. Centrifuge Centrifigation16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Re-insert column in collection tube. Add Amount200 µL of wash buffer 1. Centrifuge Centrifigation16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Add Amount400 µL of wash buffer 2. Centrifuge Centrifigation16000 x g, 00:01:00 . Discard the flow-through in the chemical waste container.

1m
Replace the spin column in the (now empty) collection tube. Centrifuge Centrifigation16000 x g, 00:01:00

Note
This clears out any left-over ethanol.

1m
Elute
Elute
2m
2m
Transfer the column to a clean 1.7 ml microcentrifuge tube. Be careful: make sure the tip of the column doesn't come into contact with the flow-through.
Add Amount30 µL of elution buffer to the center of the silica matrix. Wait Duration00:01:00 , then centrifuge Centrifigation16000 x g, 00:01:00

2m
Measure the concentration and purity of your DNA on the NanoDrop.