Looking for quick and dependable purification of your amplicon? Easily clean up your PCR product , either from an agarose gel or directly from the reaction. Plan on using your amplicon for cloning? Seamlessly insert your PCR product into any vector, at any site of linearization using ligation-independent cloning.
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We use cookies to improve your browsing experience and provide meaningful content. Read our cookie policy. New products. Contact Sales. Overview Results Methods. Overview Colony PCR is a method used to screen for plasmids containing a desired insert directly from bacterial colonies without the need for culturing or plasmid purification steps.
Figure 2. Related products Cat. Notice to purchaser Our products are to be used for Research Use Only. Documents Components Image Data Resources. Documents Image Data. PCR learning center. The more background, the more colonies you will need to screen. Saving clones for later culture : At this point, you will want to hang onto your clones for later use. There are a few ways you can do this. If you are going to complete your colony PCR analysis in the same day, you can save the leftover bacteria-water suspension and use them to start cultures of your positive clones.
If you want to store your clones longer term, just streak the colonies on an LB plate. You can use this plate to start liquid cultures.
Lastly, you can start small overnight liquid cultures with the clones you pick and only mini-prep the positive ones. Regardless of which method you choose, make sure to use the appropriate antibiotic for selection. Lysing bacteria and setting up PCR reactions : The remaining bacteria-water suspension will serve as the template for your PCR reaction.
You just need to lyse the bacteria to release the plasmid DNA by either briefly boiling the sample before use or by directly adding a small volume of the sample to the PCR reaction. The bacteria will be lysed during the initial heating step of the PCR reaction. A standard Taq polymerase is sufficient. Controls: Controls can make or break an experiment.
The best controls for a colony PCR are the same ones used to verify if the colony PCR primers work in the first place: the backbone vector with and without an insert. These controls are quick references you can use when you run your PCR products out on a gel to determine if the colonies contain an insert.
They also serve as controls for your PCR reaction. Running a no template control PCR reaction for detecting contamination is also a good idea. Make sure to use an appropriate molecular weight standard for reference and to add a loading dye with glycerol to your samples before pipetting them onto the gel.
The figure above summarizes generalized expected results for the three primers previously described. After identifying a few positive clones, the last step is to mini-prep these clones and submit the plasmids for Sanger sequencing. Sequencing allows you to confirm the sequence of the insert, insert orientation, and the sequences of the junctions between the plasmid and insert DNA.
There are lots of different cloning strategies , but regardless of which is your favorite, colony PCR is a useful tool to have in your molecular biology tool kit. Don't pick too large of a colony. Too many bacteria can inhibit your PCR reaction or cause non-specific products to show up on your gel. Beware of false positives. Make sure to submit multiple positive clones for sequencing to verify the insert sequence before proceeding with your experiment.
Shorter amplicons tend to be better. Shorter amplicons make for shorter PCR programs and are more likely to work in a PCR reaction that has bacterial debris. Use a positive control.
A good positive control is bacteria transformed with the same backbone plasmid. Use a negative control strain. A good negative control strain is an untransformed culture of the same strain of bacteria you used for cloning. This type of control is especially important for insert-specific primers.
If your negative control amplifies a product of the expected size, you know the genome of your bacteria already contains your target sequence. Add Comment. Addgene is a nonprofit plasmid repository.
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