Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into your functional experiments! Whether you’ve cloned the plasmid yourself or obtained it from a colleague down the hall, it is always a good idea to take some time to confirm that you are working with the correct construct, and verify that the plasmid you received matches the expected sequence. Here at Addgene, we process all of the plasmids we distribute for quality control purposes in order to confirm the integrity of the DNA.
Read on to learn more about our two recommended methods for plasmid DNA verification: sequencing and diagnosic restriction digest.
Sequencing determines the precise order of nucleotides within the DNA molecule, in this case a plasmid. To get started, you will first need to design and synthesize primers that perfectly compliment your plasmid sequence. We recommend starting with a backbone-specific primer that will sequence over the Multiple Cloning Site (MCS) and into YGOI. This way you can avoid designing multiple primers to verify unique genes inserted into the same backbone. Addgene has curated a comprehensive vector database that will help you find reference sequence for many commonly used backbones, as well as the specific primers used to confirm their integrity. You can also find a list of our most commonly used sequencing primers at the following link: http://www.addgene.org/mol_bio_reference/sequencing_primers/. It usually takes a couple of days to receive results after submitting your sample to a sequencing core (depending on the core facility and services available at your institution); however, it will save you time in the long run knowing that you are working with the correct plasmid. Please see our blog post on analyzing and troubleshooting sequencing results for additional tips.
Diagnostic Restriction Digest
Diagnostic digests can be used to confirm the relative structure of the plasmid based on the predicted sizes and organization of different features within the plasmid. One benefit of restriction analysis is that it can be used successfully without actually having full plasmid sequence available to you. This method is relatively quick and can be done right in your lab in less than a day (as long as you have purified DNA). Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with the selected endonucleases which cleave the DNA molecules at specific sites; and 2) running the reaction on an agarose gel to determine the relative sizes of the resulting DNA fragments.
The most common way of utilizing a restriction digest is to confirm the presence of an insert in a particular vector by excising it from the backbone. This is accomplished by using a combination of specific endonucleases that flank the insert. You will need to know both the approximate size of the vector backbone as well as the predicted size of the insert. You can search NCBI for YGOI to find the particular reference sequence if necessary.
The example plasmid on the right has a total size of 7.3kb, with the insert comprising 1.2kb of the total. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose gel. The resulting gel image includes a 1kb ladder (lane 1) that has bands ranging from about 500bp to 10kb, with the 3.0kb fragment having increased intensity to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed and nicked marked with asterisks (*). The digested samples in the last 3 lanes include HindIII and BamHI alone resulting in a single band of the full size of the plasmid, ~7.3kb, and one double digest with HindIII and BamHI together, matching the backbone size of about 6kb and the released insert at about 1.2kb (red box). The results on the gel match the predicted sizes inferred from the plasmid information.
Watch this video for a quick overview of how to analyze a restriction digest:
Restriction Digest Tips and Tricks:
The following tips will make it easier for you to obtain a useful and informative diagnostic restriction digest.
For your digest:
- Try choosing unique enzymes. Enzymes that only cut once allow you to more easily and accurately visualize the full size of your construct.
- Consider buffer and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer’s manual for the optimal working conditions for each enzyme.
- Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity may be blocked. If you have to use these enzymes for your digest, you will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110.
- Avoid star activity. Some endonucleases (for example BamHI) are capable of cleaving sequences which are similar, but not identical, to their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases and/or supply custom buffers as means to avoid this issue.
For your gel:
- Add ethidium bromide (EtBr) to your gel before pouring it. EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light, and adding it to your gel will save time!
- Don’t forget to add loading buffer to you digest reactions before loading them. The glycerol in the buffer will make sure your sample settles in the gel well and the dyes provide a visual reference point so you can easily assess how far the gel has run. Bonus: The dyes also run at predicted sizes so you can estimate how far down the gel your bands have travelled based on the dye!
- Always run a ladder. Ladders allow you to interpret the bands that you get in your sample lanes. Choose your ladder based on the expected band sizes.
- Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see 3 bands. This is due to the fact that the circular DNA takes on several conformations the most abundant being: supercoiled, relaxed and nicked. If your digest lanes look like your uncut lane then there is something wrong!
- Quantify your DNA. Loading too much DNA will make it difficult to obtain crisp bands and analyze the results. Bonus: knowing how much DNA you have loaded in each well will allow you to approximate the DNA mass of comparably intense samples of similar size.
- Run the gel at 80-150V until you have good separation between your bands. Stopping the gel when the bromophenol blue dye line is approximately 75-80% of the way down the gel will ensure you keep smaller bands from running off; however, you may need to run the gel for longer to achieve good separation of larger DNA fragments.
I hope these tips demonstrate that plasmid verification is not just necessary but also an easy process. Please vist Addgene's resource for plasmid verification to find additional tips and detailed protocols on topics such as how to set up your digests and pouring/running a DNA gel.
More Plasmid Eductional Resources: