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

[@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 ; TH-cam: th-cam.com/video/1fukzAdTvrk/w-d-xo.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: th-cam.com/video/9UlP25YVVLE/w-d-xo.html   
 
more on minipreps: bit.ly/minipreps ; TH-cam: th-cam.com/video/fKf-g5oNvLY/w-d-xo.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

[@надя-ф5и9б]

THANK YOU 🫶

[@mappermania6816]

thanks for all the info!