If you plan to work with purified DNA in the lab, it’s likely that you will use a commercial DNA extraction kit to isolate and purify your DNA of interest. With so many types of kits available, it can be a major challenge to choose the best one to use when working with an unfamiliar type of sample! In this article, I’ll review the considerations that must be taken when choosing a DNA isolation kit for your experiments.
What’s in a Kit, Anyway?
Most DNA isolation kits these days follow the same basic steps: lysis, purification onto a column, washing away impurities, and extracting off the column. However, kits vary widely in how they accomplish each step.
What Kind of Sample Are You Working With?
Different kits exist for different DNA sources, ranging from cell cultures grown in the lab, clinical samples, soil samples, and even fingerprints! Many kits have been developed for specialty applications. For instance, did you know that humic acids in soil or the hemoglobin in blood can contaminate purified DNA and lead to interference with downstream DNA experiments like PCR? Luckily, there are kits that specifically remove these components prior to the purification step.
Other times, you may be looking to purify DNA from a PCR or agarose gel. In that case, there are many ways to improve recovery from agarose gel purifications.
Genomic vs Plasmid DNA Isolations
Generally speaking, plasmid DNA isolation must be gentler than genomic DNA isolation. This is because the lysis step when targeting plasmid DNA requires that the chromosomal DNA remains bound to the cellular protein fraction to keep it from contaminating the final sample. Kits exist for both types of DNA recovery, and there are even kits that can purify both genomic DNA and total RNA.
Cell Line or Organism
Arguably the most important variable in selecting a DNA extraction kit is the organism you are working with.
- Bacteria: Many kits are available with varying components for bacterial DNA isolation. The main differences between these kits is how cells are lysed, since some bacteria are more challenging to break open than others. For instance, bacteria that form biofilms may require more intensive lysis techniques than the traditional detergent-based method.
- Animal tissue: Kits intended for animal tissue DNA isolation often have gentler lysis techniques since most animal cells do not have a cell wall like bacterial ones. However, the challenge behind animal tissue DNA extraction often lies in selecting an appropriate tissue homogenization method. In addition, many kits for animal DNA purification do not use phenol/chloroform extraction or ethanol for the precipitation step to reduce the risk of damaging the DNA.
- Plants: When it comes to plant DNA isolation, a few other considerations must be taken into account. Plants often have sugars that are not separated during the purification step in other kits, so detergents that selectively bind to DNA and precipitate out of solution are often used followed by dissolving the detergent in a highly concentrated salt solution and extracting DNA afterward. There are several other techniques to remove contaminants from plant cell samples that you can read more about here!
- Blood: Thanks to many recent advances in technology, there are multiple lysis techniques and extraction methodologies used in blood DNA isolation kits to choose from. What you will be doing with DNA isolated from blood will largely dictate the type of lysis and extraction you use. Regardless of the application, be sure to choose a kit that will provide pure, intact, double-stranded and concentrated DNA for the best results.
How Much DNA Do You Need?
Most companies that sell DNA isolation kits offer kits with similar components that can be scaled depending on the volume of sample being processed. Here’s a rundown of recommended sample volume for DNA isolation kits based on E. coli– as you can see, there’s a wide range available!
- Miniprep: >15 mL
- Midiprep: 15-25 mL
- Maxiprep: 100-200 mL
- Megaprep: 500-2,500 mL
- Gigaprep: A whopping 2.5-5 L!
For specialized applications, there may be less selection when it comes to the quantity of culture or tissue sample being processed. Reach out to several companies to see what is available!
Your Time Is Valuable: Throughput Considerations
It doesn’t make sense to use column minipreps when you are processing more than 50 samples at a time. Consider high-throughput DNA extraction kits that can be run in a 96-well format to stay sane during experiments where DNA from many samples must be processed at once!
Last, but Not Least…
After going through this list, it’s clear that kits come in all shapes and sizes. A common thread, however, is that the kit you use should result in clean and concentrated DNA. You should periodically evaluate the quantity and quality of DNA extracts to ensure the kit you have chosen works well for your application. If you are struggling with a particular method or kit, be sure to check out the plethora of DNA isolation troubleshooting articles on Bitesize Bio or call the manufacturer. Keep at it and you will get your DNA of interest in no time!
What types of kits have you used in the lab? Have you extracted DNA from a particularly interesting source? Tell us in the comments below!Image Credit: andy johnston