May 12, 2026
Monarch Spin Plasmid Miniprep (NEB)
- Owen Butler1,
- Raven Coffer1
- 1DAMP Lab
- DAMP Lab
Protocol Citation: Owen Butler, Raven Coffer 2026. Monarch Spin Plasmid Miniprep (NEB) . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld46yxl5b/v1
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: March 16, 2026
Last Modified: May 12, 2026
Protocol Integer ID: 313411
Keywords: miniprep, DNA Extraction, spin plasmid miniprep kit from neb, monarch spin plasmid miniprep, spin plasmid miniprep kit, dna from overnight culture, goal of this protocol, dna, neb, protocol
Abstract
This protocol uses the Monarch‱ Spin Plasmid Miniprep Kit from NEB (Cat # T1110). The protocol has been adapted from the NEB recommended protocol using centrifugation. The goal of this protocol is to extract plasmid DNA from overnight cultures of E. coli.
Guidelines
- All work should be performed close to the bunsen burner.
- All centrifugation steps are performed at 16,000 rcf.
- NFW should be heated to 50 C prior to elution
Materials
- Overnight cultures
- Culture media
- LB, M9, etc
- Clear, round-bottom 96w plate
- Eppendorf tubes (1.5 mL)
- Bunsen burner
- Pipette / tips
- Centrifuge
- Plate reader
- Bleach (10%)
- Nuclease Free Water
- Bleach waste beaker
- Ethanol waste beaker
Before start
1. Check to see if RNase A has been added to Buffer B1 for a final concentration of 100 µg/mL.
- For T1110G (10-prep) kit, add 12 µl of Monarch RNase A to Monarch Buffer B1.
- For T1110S (50-prep) kit, add 60 μl of Monarch RNase A to Monarch Buffer B1.
- For T1110L (250-prep) kit, add 285 μl of Monarch RNase A to Monarch Buffer B1.
2. Check to see if isopropanol has been added to Buffer BZ (0.43 volumes of isopropanol per volume of Monarch Buffer BZ).
- For T1110G (10-prep) kit, add 1.5 ml of isopropanol to Monarch Buffer BZ.
- For T1110S (50-prep) kit, add 3.6 ml of isopropanol to Monarch Buffer BZ.
- For T1110L (250-prep) kit, add 18 ml of isopropanol to Monarch Buffer BZ.
3. Check to see if ethanol has been added to Buffer WZ (4 volumes of ≥ 95% ethanol per volume of Monarch Buffer WZ).
- For T1110G (10-prep) kit, add 4 ml of ethanol to Monarch Buffer WZ.
- For T1110S (50-prep) kit, add 20 ml of ethanol to Monarch Buffer WZ.
- For T1110L (250-prep) kit, add 104 ml of ethanol to Monarch Buffer WZ.
Set Up
3m
Preparation
Wipe benches and pipettes with DNA-Away and then RNAse-Away. Afterwards, wipe with 70% ethanol. Light the bunsen burner & work closely to it while performing the Validation of Overnight Culture steps.
3m
Validation of Overnight Culture
24m
OD600 Measurement of Overnight Cultures
Obtain a clear round-bottom 96w plate. Download the attached excel sheet. Record the specific plate map that will be used for each run on the excel sheet.
OD600_analysis.xlsx11KB The plate map below is an example for 2 samples.
| 1 | 2 | 3 | |
| A | BLANK | BLANK | |
| B | SAMPLE 1 | SAMPLE 1 | |
| C | SAMPLE 2 | SAMPLE 2 | |
| D |
3m
In the clear round-bottom 96w plate, add 100 µL of the appropriate media in duplicate in the designated wells from the plate map above. These are the BLANKS.
Note
The blanks are used to account for the background absorbance due to the media in which the cells are grown.
2m
In the clear round-bottom 96w plate, add 90 µL of appropriate media and 10 µL of each overnight culture (pipette to mix before transferring culture) in duplicate into the designated wells, to make 1:10 dilutions. Ensure to mix the wells thoroughly once added.
Note
Save overnight cultures for the following steps.
Read OD600 using the BioTek Synergy H1 microplate reader. Copy the blanks and the OD values into the excel sheet downloaded in a previous step. The sheet will perform the following calculations to correct the sample measurements.
ODcorrected = (ODsample −
5m
Save and file the excel sheet in the appropriate location.
1m
Once finished reading, add 10% bleach to each sample well of the 96w plate. After 10 minutes, empty the plate down the drain with water. Discard the plate in a biohazard box.
10m
If the cultures are sufficiently grown (ODcorrected > 1), proceed. Extinguish the bunsen burner.
Miniprep
36m
Isolating and Resuspending Culture
Discard the supernatant into a 10% bleach solution.
5m
Resuspend the pellet in 200 µL of Buffer B1. Gently pipette to mix. Transfer to a pre-labeled 1.5 mL eppendorf tube.
5m
Lysis & Neutralization
Add 200 µL of Buffer B2, gently invert the tube 10 times (do not vortex), and incubate at room temperature for 00:01:00 . Color should change to dark pink, and solution will become transparent and viscous.
Note
Avoid incubating longer than 1 minute to prevent irreversible plasmid denaturation
1m
Add 400 µL of Buffer B3, gently invert the tube until neutralized, and incubate at room temperature for 00:02:00 . The color should be uniformly yellow when neutralized.
2m
Begin pre-heating Elution Buffer or NFW to 50 °C in a bead-bath or incubator for the elution step later on.
DNA Binding & Wash
Centrifuge lysate at 16000 x g, 00:05:00 .
Note
Pellet should be compact and adhered to the side of the tube. If it is not strongly adhered to the side of the tube, spin again at 16000 x g, 00:05:00 .
Pellet should be compact; if it is not strongly adhered to the side of the tube.
5m
Carefully transfer 800 µL supernatant to the spin column and centrifuge at 16000 x g, 00:01:00 .
Note
Tilt the eppendorf tube away from the pellet and carefully pipette up the supernatant, ensuring the pipette tip does not touch the pellet or any debris stuck to the side of the tube.
1m
Discard flow-through in the collection tube into a waste beaker labeled "Ethanol waste."
Dab the bottom of the collection tube on a KimWipe to absorb any excess flow-through.
Re-insert the column into the same collection tube and add 200 µL of Buffer BZ. Centrifuge for for 16000 x g, 00:01:00 .
1m
Discard flow-through in the collection tube into the waste beaker labeled "Ethanol waste."
Dab the bottom of the collection tube on a KimWipe to absorb any excess flow-through.
Add 400 µL of Buffer WZ and centrifuge for 16000 x g, 00:01:00 .
1m
Place the filter back into the same collection tube. Spin for 16000 x g, 00:01:00 .
1m
Transfer column to a clean, pre-labeled 1.5 mL eppendorf tube.
Note
Use care to ensure that the tip of the column does not come into contact with the flow-through in the collection tube.
With the snap-cap open, let ethanol evaporate for 00:02:00 from the column while sitting in the 1.5mL eppendorf tube.
2m
Elution
Add 45 µL pre-heated to 50 °C . Elution Buffer or NFW to the center of the column's matrix. Incubate for 00:05:00 at 37 °C . Spin for 16000 x g, 00:01:00 .
6m
Transfer the flow-through now in the 1.5mL eppendorf tube back into the column's matrix to maximize yield. Incubate for 00:03:00 at 37 °C . Spin for 16000 x g, 00:01:00 .
Note
Although this step may decrease final elute volume, it can increase yield.
4m
For short-term storage ( < 1 day), store at 4 °C .
For long-term storage ( > 1 day), store at -20 °C .
Validation of Miniprep Yield
10m
Nanodrop
10m
Clean Up
12m
Waste Disposal
Add 10% bleach solution to remaining overnight cultures. Wait 00:30:00 . Pour down the drain with water. Dispose of culture tubes in biohazard.
Empty benchtop sharps container.
1m
Empty ethanol beaker into the ethanol waste collection site.
1m
Acknowledgements
Drafted by: Raven Coffer
Uploaded by: Luciana Lozano
Edited by: Raven Coffer, Owen Butler, Kristen Sheldon, Lena Landaverde