CoPurifying plasmid DNA from bacterial cultures is a core protocol in molecular biology. And whilekit-free plasmid purification is always an option, let’s be honest — kits are quick, easy, and don’t involve phenol-chloroform.
The default option is to use the kit that is already available in your lab or the one you are most familiar with. And, hey — if something is working for you, then there is no reason to change it. But what if a kit isn’t working for you, or if you don’t have an old standby to fall back to? We have outlined some important considerations to help you choose the plasmid prep kit that is right for you.
What type of plasmid prep do you need?
As we covered in a previous post on plasmid preps, not all plasmid preps are the same. Your downstream application will dictate how much DNA you need to recover and the level of quality required. Some applications require very little DNA and you can get away with slightly lower quality preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based on their relative quality and quantity.
- Transformation Grade DNA (low quality/low quantity) – Bacterial transformation protocols require such small amounts of DNA (often on the picogram scale) that most quality issues are negligible. That said, “low” is a relative term — even armed with the highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded.
- Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning experiment with a relatively small amount of DNA (in the nanograms to micrograms scale depending on the experiment), but issues with DNA quality can inhibit important reactions like PCR or restriction digests.
- Transfection Grade DNA (high quality/high quantity) – Depending on the experiment, mammalian cell transfections can require quite large amounts of DNA (easily in the micrograms scale and up). But perhaps more importantly, the DNA needs to be free of contaminants, such as bacterial endotoxins, that could affect your culture’s viability.
- Avoid Low Quality/High Quantity – We recommend avoiding a High Quantity/Low Quality mindset for any applications.
DNA quantity is fairly self explanatory and achieved by performing different sized preps, such as miniprep, midiprep, etc. (more on prep sizes below). But what do we mean by DNA quality? There are a few different issues that can arise during the plasmid purification process. One is the possibility of damaging the plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a little overzealous with your pipetting. Another is the possibility of carrying over contaminants from the plasmid prep process into the final plasmid eluate. Possible contaminants can sneak in from almost any step of the process and include things like genomic DNA, RNA, proteins, salts, ethanol, or endotoxins. The most common method for checking both quantity and quality of your plasmid prep is by spectrophotometry.
Achieving the highest-quality DNA requires taking care at each step, but some purification kits make it easier than others to prevent quality issues. For example, some kits reduce the risk of ethanol carry-over into the eluate, while other kits are guaranteed to yield endotoxin-free DNA. Once you know the type of DNA prep you need, you can better assess which kit to use for your experiment.
Bead-based or column-based kits — which to choose?
Plasmid purification protocols can be broken into a few broad stages: lyse the bacteria harboring your plasmid; separate plasmid DNA from unwanted cellular “debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing cellular debris look very similar. However, keep in mind that kit components (i.e. buffers, columns, or beads) are designed to work together — no mixing and matching!
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For the clean-up stage, plasmid purification kits typically use one of two common approaches — columns or magnetic beads. Both approaches involve binding the plasmid DNA from your cleared lysate to a substrate, washing away left-over debris that carried over from the precipitation stage, and then eluting the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed in a column and you flow lysate and buffers through the matrix, usually by centrifugation or vacuum. In a bead-based approach, you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash and elution steps.
Both bead- and column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications tend to be easier to automate and use in high-throughput settings, but when it comes to low-throughput applications, the choice is largely a matter of preference.
Which size kit to use?
Obtaining higher quantities of DNA generally means starting with higher volumes of bacterial culture. Plasmid purification kits are designed to accommodate specific volume ranges of culture as their input (Table 1). When you feel like you need just a bit higher DNA yield, but not enough to go up a kit size, it may be tempting to try increasing your input to increase your output. But, exceeding a kit’s capacity can actually reduce your ultimate plasmid yield.
Take your average column-based miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of buffers used to ensure proper lysis, but you will still face column capacity limitations since the columns can be “clogged” by excess material, leading to less DNA in your final eluate. Bead-based kits can be more flexible in this situation as you can simply add more beads to your sample. But, you’ll need to ensure you have an appropriate amount of beads and a suitable magnet as you scale up.
Table 1: Standard Prep Kit Capacities*
*Can vary from kit to kit, check the manufacturer’s documentation
**Varies by plasmid copy number
Pro tip! If you are having trouble getting sufficient plasmid DNA, you may need to adjust your starting culture conditions rather than your kit.
Do you want eco-friendly options?
It’s no secret that labs produce a lot of waste. If you are looking to reduce your lab’s environmental impact, check out Addgene’s blog post on bringing sustainable practices for the lab. Plastic consumables (including pipette tips, plates, and yes those purification columns we’ve been talking about!) are a significant source of waste in laboratories. To help combat this, some manufacturers have designed eco-friendly DNA purification kits by reducing the amount of plastic and packaging material or by incorporating recycled paper and plastic.
Aside from the more obvious swaps like using smaller bottles or recycled cardboard packaging, there are some less obvious improvements that reduce the environmental impact. Using non-silica-based purification protocols requires less plastic and avoids the use of more hazardous reagents like guanidine salts. Using concentrated buffers reduces plastic waste from bottles and the overall weight of the package during shipping. Of the kits reviewed here, NEB’s Monarch®, QIAGEN’s QIAwave, and Milllipore Sigma’s GenElute™ kits were all specifically designed with environmental sustainability in mind (Table 2).
Table 2: Sustainability features highlighted in plasmid purification kits
What are all these other kit types?
In addition to standard purification kits, there’s a range of specialty kits available for plasmid purification. Kits can be optimized based on the plasmid size, cell model, or overall throughput needed.
- Large constructs – Large plasmids, cosmids, or BACs, can be tricky to purify due to their size. Some manufacturers offer kits specifically optimized for purification of these large plasmids, while others have suggested modifications to their normal kit protocols.
- Yeast plasmids – If you’re extracting plasmids from yeast, instead of bacteria, a few manufacturers (such as Zymo and TakaraBio) offer yeast-plasmid specific kits.
- Medium/High-throughput – Many manufacturers offer bead- and column-based 96-well plate options for medium-throughput applications. For high-throughput options, several manufacturers offer automation-friendly kits and/or automation systems that can enable you to run thousands of reactions with relatively little work after the initial setup. (Addgene actually uses some of these — if you’re curious check out our blogpost on lab automation here!)
- Rapid Isolation – If you’re in a hurry, several manufacturers also offer kits with speedy protocols — anywhere from eight to sixteen minutes. (These are mostly miniprep kits! Larger kits generally take longer.)
Make sure to take a look at the manufacturer’s kit pages as well! Some kits that may not be explicitly labeled as “yeast”, “transfection-grade”, or “large plasmid” may be suitable for these purposes with modified protocols.
*Note that this blog post only covers plasmid purification kits — there are many more kits out there for extracting DNA from tissues, blood, virus, or other samples, for PCR and gel cleanup, and for extracting RNA! (That’s a blog for another time, however!)
Where can you look for help?
All manufacturers provide specs, protocols, and quality control information, but the level of detail can vary. If you’re new to using purification kits, or if you’re struggling with a purification, reading the full instruction manual will help explain the rationale behind each step and provide troubleshooting advice. In these cases, it can be helpful to select a kit with easy-to-follow instructions and extensive references for troubleshooting. If you need a high level of consistency between lots, then reviewing their quality assurance documentation can help you select a kit.
So...which kit should you pick?
To make it easier to compare kits at a quick glance, Table 3 below contains details regarding kits from a variety of manufacturers. It also includes a rough comparison of list price between these manufacturers, as price is a large consideration for most labs. Many institutions have preferred vendors which may reduce costs for certain kits, so be sure to check with your lab manager or ordering department for these.
While budget is an important consideration, you do want to make sure you’re getting the best bang for your buck. If you’re unsure if a kit is a good option for you, some manufacturers offer trial or sample sizes of their kits, which you can use to test or compare kits before deciding which one to buy.
Table 3: Comparison of popular plasmid purification kit options
While we’ve included a sample of some commonly used kits, please note that Table 3 is certainly not comprehensive — there are many other vendors and kits out there as well. The correct kit for your lab is dependent on many different factors as we detailed above, so be sure to pick what kit works best for you! Happy purifying!
This blog post was written by Ashley Waldron, Hannah Dotson, and Nyla Naim.
The company is the world’s best DNA Methylation Kits supplier. We are your one-stop shop for all needs. Our staff are highly-specialized and will help you find the product you need.
Resources
Additional resources on the Addgene blog
- Plasmid Preps: Different Purity, Different Quantities, Different Uses
- Plasmids 101: Optimizing Plasmid Yields
- Bringing Sustainable Practices to the Lab: Easy Action Items
Additional resources on addgene.org
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