Expert Tips & Tricks for Plasmid DNA Purification Using Miniprep Kits

Purifying DNA plasmids using a miniprep kit may seem like a straightforward task, but what happens when things don’t go according to plan? Unexpected issues can arise, from low yields to impure samples. In this blog post, we’ve asked our scientists what some of the most essential tips and tricks are to help you face any plasmid purification challenges that can occur. By troubleshooting common problems, you can ensure your plasmid purification process is as efficient and reliable as possible. Read on for expert insights and practical advice for mastering DNA plasmid purification with miniprep kits.

A miniprep kit (such as the Monarch^® Spin Plasmid Miniprep Kit, (NEB #T1110) generally follows a similar set of steps, so we’re dividing this post into tips and tricks for each step: Sample Preparation (Growing bacterial culture), Resuspend, Lyse, Neutralize, Bind, Wash, and Elute.

 

(1) SAMPLE PREPARATION (Growing bacterial culture)

Grow your bacterial culture overnight to obtain a sufficient quantity of cells containing the plasmid DNA.

• Use a fresh growth media plate and antibiotic when growing colonies to start your culture.
• Inoculate growth media from a single colony.
• Ensure you use the proper antibiotic at the correct concentration to maintain selection during the growth of the culture.
• Harvest the culture during the transition from logarithmic growth to stationary phase (typically 12–16 hours for growth in LB medium) to ensure the greatest quantity of plasmid DNA is present, and little cell lysis has occurred.

• Don’t select satellite colonies when inoculating the culture, as these may not contain the desired plasmid.
• Don't sub-culture ampicillin-maintained cultures, as this can lead to the depletion of antibiotics by secreted β-lactamase.
• Don't allow the culture to grow for extended periods beyond the recommended time, as this can lead to cell lysis and low DNA yield.
• Don't use E. coli strains like HB101 and the JM series if you can avoid them, as they have high levels of endogenous endonuclease that can degrade plasmid DNA.

 

 

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(2) RESUSPEND

Resuspend the bacterial pellet thoroughly in a buffer solution to prepare the cells for the lysis step.

• If you are working with a low-copy plasmid, increase the number of cells processed and scale up the buffers accordingly to accommodate the increased number of cells.
• Ensure the cell pellet is completely resuspended before adding the Lysis Buffer.
• If using the Monarch Spin Plasmid Miniprep Kit, be sure to add RNase A to Buffer B1 to avoid RNA contamination.

• Don't use more cells than recommended (up to 5 ml, equivalent to 15 OD units), as this can result in inefficient cell lysis and clogging of the matrix, which can lower DNA yield.

 

 

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(3) LYSE

Lyse the bacterial cells by adding a lysis buffer, which releases the plasmid DNA into the solution.

• Confirm the color change from light pink to dark pink and transparent during the lysis process
• Promptly move on to the neutralization step after the lysis step to prevent plasmid denaturation.
• Mix carefully by inversion after cell lysis to avoid host chromosomal DNA contamination.

• Don't extend the incubation time (ideally, no more than 2 minutes) in the presence of sodium hydroxide (in the Monarch Buffer B2 Lysis Buffer) during the lysis step, as this can separate the DNA strands and irreversibly denature the plasmid.
• Don't mix vigorously or vortex after cell lysis and before pelleting cell debris, as this can shear the host chromosomal DNA and contaminate the plasmid. Note: if your prep contains contaminating genomic DNA that was sheared during lysis, it may co-purify with the plasmid and will likely appear as a high-molecular-weight band on an agarose gel.

 

 

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(4) NEUTRALIZE

Neutralize the lysate with a neutralization buffer, allowing gDNA, proteins and other cellular debris to precipitate out, while the plasmid remains in the solution.

• If RNase A was added to the buffer in the Resuspension step, ensure the sample is well mixed and allow it to incubate for the recommended time to allow for RNA degradation. For the Monarch Spin Plasmid Miniprep Kit, incubate for 2 minutes to allow RNase to degrade RNA.
• Gently invert the sample tube enough times to ensure a complete and uniform color change to yellow during neutralization. 
• Make sure cell debris appears in abundance and is fully compacted into the pellet after centrifugation.

 

 

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(5) BIND

Bind the plasmid DNA to a silica column by passing the lysate through it, allowing the plasmid DNA to adhere to the column.

• Only transfer the supernatant to the column. Make sure the lysate is free of cellular debris before applying it to the column to avoid clogging.
• Use the correct volume of lysate recommended by the kit to ensure optimal binding of plasmid DNA.

• Do not overload the column. For the Monarch Spin Plasmid Miniprep kit, the maximum column loading volume is 800 μl. If the supernatant volume is > 800 μl, load the first 800 μl, spin through, discard flow-through and reload the remaining volume.
• Don’t hurry the binding step; insufficient contact time can lead to poor DNA recovery.

 

 

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(6) WASH

Wash the bound DNA on the column with a wash buffer to remove any remaining contaminants and impurities.

• Ensure the final wash spin time is 1 minute to enable the complete removal of the wash buffer from the column.
• Be aware that strains like HB101 and the JM series have high amounts of endogenous carbohydrates that can interfere with downstream enzymatic manipulations of plasmid DNA. To remove excessive carbohydrates, be sure to include the first wash step with the Monarch Buffer BZ to help reduce plasmid degradation, and keep the samples on ice during preparation.

• For the Monarch Spin Plasmid Miniprep Kit, don't skip any wash steps in the protocol to help remove any residual RNA, protein and other contaminants.

 

 

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(7) ELUTE

Elute the purified plasmid DNA from the column by adding an elution buffer and collecting the eluate.

• Use the recommended elution volumes and incubation times for typical plasmids in the < 15 kb range. Larger elution volumes and longer incubation times can increase the yield of DNA eluted from the column; however, the sample will be more dilute, and the processing time will increase.
• To improve the yield for larger plasmids, incubate the column at room temperature for 5 minutes or heat the elution buffer to 50°C before adding to the column.
• Add the elution buffer to the center of the matrix to ensure the matrix becomes evenly wet.
• If eluting in water instead of elution buffer, be sure the water is nuclease-free and the pH is between 7-8.5. Milli-Q™ water is often slightly acidic, requiring pH adjustment. If you are storing the DNA long-term, we recommend using the supplied DNA elution buffer, which contains 0.1 mM EDTA and can help inhibit metal-dependent nucleases.
• Store the DNA at -20°C to ensure its stability, if it will not be used immediately. Consider enzymatically removing contaminating genomic DNA using Exonuclease V (RecBCD) (NEB #M0345).

• Don't use smaller elution volumes or shorter incubation times than recommended. This can yield highly concentrated DNA, but can result in incomplete elution and low DNA yield.
• Don't store DNA in a solution containing magnesium, as this can degrade the DNA.
• Don't let the column tip contact the flow-through during the transfer to a new tube. If in doubt about ethanol carryover, re-spin the column for an additional 1 minute.

 

 

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Some Advantages of the Monarch Spin Plasmid Miniprep Kit are:

• Colored buffer to help you follow the workflow and remember which steps you’re on if you get distracted.
• Spin columns are precision-engineered to allow for low elution in as little as 30 µl.
• Designed for sustainability without compromising performance. The kit uses less plastic, flexible purchasing options, and uses sustainable and recyclable packaging.
  
  

 

If you’re having any problems with your DNA plasmid purifications, our technical support team is always available to help.
You can find a full list of FAQs for the Monarch Spin Plasmid Miniprep Kit here

We also have Monarch kits, protocols and technical support for:
- Monarch PCR & DNA Cleanup Kit (5 µg) (columns also sold separately) (NEB #T1030)
- Monarch DNA Gel Extraction Kit (columns also sold separately) (NEB #T1020)
- Monarch Genomic DNA Purification Kit (columns also sold separately) (NEB #T3010)
- Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050)
- Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060)

Monarch Nucleic Acid Purification Kits