Colony PCR - a quickish way to see if your cloning probably worked: a time, thumb, & resource-saver!

Colony PCR… This week I introduced a colleague to the wonders of this time, thumb, & resource-saver! In molecular cloning, we can take genetic recipe for making a protein and stick that recipe into a circular piece of DNA called a plasmid. That plasmid serves as a vector or “vehicle” for getting (and keeping) the protein instructions in bacterial cells. But before we try to get cells to make the protein, we want to make sure that the recipe got into the plasmid okay and there aren’t any typos. A technique called colony PCR can quickly tell us (with not purification required) if our recipe likely got in there before we waste time and resources purifying the plasmid and sending it for sequencing if it’s bad.⁣ Here’s a bit about how it works… note - text from past posts
 
blog form: bit.ly/colony_PCR⁣  
 
Polymerase Chain Reaction (PCR) is a way to amplify (make lots of copies of) short stretches of DNA from longer pieces of double-stranded (ds) DNA we call the TEMPLATE. We choose what region to copy by designing short pieces of DNA called PRIMERS to bookend the start & stop of this region (1 per strand) so that a protein called DNA POLYMERASE (DNA Pol) can copy each strand.. more here: bit.ly/pcrtrain  
 
But where does the template itself come from? That depends. In colony PCR it’s typically a molecularly-cloned plasmid. In the process of MOLECULAR CLONING bit.ly/molecularcloningguide I put (edited) gene for a protein I want to study from one template and put that gene INSERT into a circular piece of DNA called a PLASMID VECTOR that has the “bells and whistles” I want, like “tags” to help with purification and start signals for turning the gene into protein. Then I stick this RECOMBINANT plasmid into bacterial cells so the bacteria will make more of the DNA and/or protein. 
 
But how do I know if the bacteria really have my gene in them? The plasmid vector has a selection marker - often an antibiotic resistance gene - so that if you grow the bacteria that should have it on food containing that antibiotic, only the bacteria that have the plasmid (and hence the resistance gene) are able to grow. These bacteria grow and replicate to form individual “colonies” on a bacterial plate. Each colony has lots of cells but they all have the same genetic makeup 
 
BUT this only tells you if the plasmid is inside the bacteria not if your gene is inside that plasmid. To answer this latter question, you can use PCR with cleverly designed primers. You have a few options and presence/size of the copied produces (which you can tell by agarose gel electrophoresis) can tell you different things: 
 
INSERT-SPECIFIC PRIMERS: both primers are in the INSERT (the gene you put in). This is a YES/NO for whether your insert’s present. If your gene’s not there there will be nothing for the primers to bind to → no product. But if your gene is there the primers will latch on & Pol will copy between them → product (note that by product I mean a defined, specific product, not “nonspecific products” that can come from primers binding incorrectly (mispriming) 
* tells you if your gene is present BUT NOT if your gene is where you want it… 
* advantage is that you can use this same set of primers to test for your insert in different plasmids 
 
VECTOR-SPECIFIC PRIMERS: both primers are in the VECTOR, straddling the insertion site. As long as the plasmid’s present, you should get some sort of product, but it’s the SIZE of the product that gives you your answer (not a simple yes/no like above) - if your insert’s not in the vector the product will be really short but if your insert’s in there, the product should be bigger (that short length PLUS the length of your insert) 
* tells you if your gene (or something of that same size) is present IN YOUR VECTOR 
* useful because you can use the same pair of primers to test different constructs since the primers are specific for the vector not the insert 
* does NOT tell you whether your insert is inserted in the correct direction. for that you can use 
 
ORIENTATION-SPECIFIC PRIMERS: one primer is in the insert & the other is in the vector 
* you’ll only get a defined product if your gene if facing the right way (not put in backwards so that the “start making protein here” message on the plasmid is next to the “stop making protein here” message on the DNA 
* tells you 1) is your plasmid present 2) is your gene present 3) is your gene in your plasmid and 4) is your gene “backwards” 
* downside is you have to design a specific primer 
 
finished in comments

[@user-uf9zk3uv6e]

THANK YOU 🫶

[@thebumblingbiochemist]

So we can use PCR as a secondary “screen” when cloning, but we still haven’t answered the question of how we get the DNA to screen. You can purify plasmid DNA out of bacteria - often using easy-to-use “mini prep kits” - they’re easy to use but if you have lots of bacteria to test, you don’t want to waste time purifying something “useless” so you can skip the purification (for now) and add a teeny bit of the whole bacterial cells into your PCR mix. 
 
Just barely touch the colony with a sterile toothpick or pipet tip & swirl it around a bit in your PCR mix. (alternatively, you can resuspend a bit of it (pipet it up in down in some water) and add some of this to the PCR mix. 
 
When the reaction heats up to MELT the DNA (separate the strands) it also LYSES the cells (breaks them open) so that the DNA “spills out” and DNA Pol can latch on. 
 
If you get a positive result, you can then go ahead and grow up more of that colony and purify it.  
 
Another “quick check” is an analytical restriction digest - more here: bit.ly/analytical_digest ; RUclips: ruclips.net/video/1fukzAdTvrk/видео.html   
but the basic idea is that you cut out, within, etc., the part of your plasmid that should contain your gene. Then you see how many & how big those pieces are (with agarose gel electrophoresis). If your gene is there the piece will be much bigger than if it’s not there and/or depending on where your cut sites are you will get more pieces. And while you can’t tell exactly how many DNA letters are there, you get an idea whether you’re in the right ballpark.  
 
BUT - with either of these methods, you still don’t know if there are any typos! (is the sequence correct?) Both restriction enzymes and colony PCR primers only require that the short stretches of DNA they recognize are there & typo-free but that’s like seeing that one word in a document is spelled correctly and then taking that as proof you didn’t make any typos anywhere else in the document.  
 
For definitive evidence, you turn to DNA sequencing. Using sequencing primers is similar in setup and concept to vector-specific colony PCR - use 1 primer that matches a sequence upstream of your gene and one downstream. But, unlike in colony PCR, where you have both primers in the same reaction, for the sequencing reactions you do the reactions separately. Instead of focusing on making tons of copies, you focus on reading carefully - you read out the sequence as you add each base. Instead of adding both primers in the same reaction, it’s one at a time, so instead of making double-stranded (ds) copies of a defined region of DNA, you start making a copy of a single strand and you “stalk it” as it works more here: bit.ly/sequenceclones  
 
note: methods like blue-white screening can help you triage which clones to check. Basically, you do your cloning so that your insert is inserted into a gene the bacteria need in order to make a blue product. If your insert (or at least something) gets in there, it disrupts that gene, so the cells can’t make the blue product and those colonies look like normal colonies, not blue. This doesn’t tell you about the size of the thing that got in, so it tells you less than the PCR and restriction cloning, but it can be used to choose which colonies to test.  
 
more on blue-white screening: blog: bit.ly/bluewhitescreening ; video: ruclips.net/video/9UlP25YVVLE/видео.html   
 
more on minipreps: bit.ly/minipreps ; RUclips: ruclips.net/video/fKf-g5oNvLY/видео.html   
 
more on clone-checking: bit.ly/colonychecking  
  
more on DNA sequencing: bit.ly/DNAsequencingmethods & bit.ly/sequenceclones & bit.ly/sequencetermstools   
     
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@mappermania6816]

thanks for all the info!