Methods to verify your gene of interest was successfully cloned
Your experiment is off to a great start and you are approaching the end of your molecular cloning workflow. Things have gone well, but how do you know that all the steps have resulted in the correct recombinant clones? Before you continue with your experiment, colonies must be screened for positive results. Pick your favorite method from these 5 ways to verify that your gene of interest was successfully cloned – the classic way, the powerful way, the precise way, the quick way, or the most accurate way.
Method #1: A classic way
Blue-white screening is a negative selection system using bacterial lactose metabolism as an indicator of successful cloning. Across the vector’s cloning site lies a DNA sequence encoding a peptide, which can be visually detected as blue colonies. When your DNA of interest is inserted into the plasmid, the gene is disrupted, and the bacterial colony becomes white in color.
Sound complicated? Here’s how it works. Take a special E. coli strain that is missing the gene sequence for α-peptide, which is a functional subunit of the β-galactosidase enzyme. Since the cloning vector contains that missing gene sequence, it can restore enzyme function to the transformed E. coli. You can see this visually with X-gal, a lactose analog that turns blue when hydrolyzed by β-galactosidase. A vector containing your DNA insert will not express α-peptide, so no β-galactosidase activity is present, and X-gal won’t be hydrolyzed.
So maybe now it seems more complicated, but the basic rule is if the DNA insert is present, the colonies will be white. If the DNA insert is not present, the colonies will be blue. Since it is possible to obtain false positives, it’s always best to follow up using one of the other methods (PCR, restriction digest, or DNA sequencing).
Method #2: A powerful way
An effective method to simplify screening is to use a positive selection system – a twist on the blue/white system mentioned above. Positive selection vectors conditionally express a lethal gene, such as a restriction enzyme that digests the genomic DNA of the bacterial host. When your DNA is successfully inserted in the plasmid, the lethal gene can no longer be expressed. As a result, only cells with recombinant plasmids are able to grow. This approach can save time and cost since the typical yield is >99% recombinant clones.
Method #3: A precise way
Using restriction enzymes to check the presence and direction of your insert is a precise and easy method for screening colonies. First, restriction mapping should be performed to identify which restriction enzymes can be used to easily identify the presence of your insert within the plasmid. After isolating a plasmid DNA from an overnight bacterial culture, digest the purified plasmid DNA from recombinant clones using restriction enzymes. Once digested, run the plasmid on an agarose gel to verify that the vector backbone and insert are of the expected sizes (Figure 1).
Figure 1. Five minute plasmid DNA digestions with M: Thermo Scientific GeneRuler Express Ladder, 1: Control, undigested plasmid DNA, 2: FastDigest EcoRI, 3: FastDigest EcoRI and FastDigest KpnI, 4: FastDigest EcoRI, FastDigest KpnI and FastDigest SmaI.
Method #4: A quick way
Colony screening with Polymerase Chain Reaction (PCR) is the most rapid initial screen to determine the presence of the DNA insert. Colony PCR involves lysing the bacteria and amplifying a portion of the plasmid with either insert-specific or vector-specific primers. If you need to determine the orientation of your insert, it is recommended to combine both types of primers for your analysis.
If selecting colony screening by PCR, make sure that your insert is shorter than 3 kb. Some PCR reagents will allow you to add a portion of your individual colony directly to a PCR master mix, with the remaining portions being used to inoculate a culture plate or liquid media with appropriate antibiotic for downstream applications.
Method #5: The most accurate way
The most accurate way to verify your recombinant colonies is by Sanger sequencing. Plasmid DNA is first isolated from an overnight bacterial culture. Once completed, the insert can be identified using sequencing primers appropriate for the selected vector. Sequencing across the entire insert is required to verify the exact sequence of the insert.
Regardless of the method you have selected to use, identification of the correct recombinant will bring your cloning workflow to a successful end!
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