Practical advice for increasing your PCR product yield

If you need more PCR product, it might be tempting to just increase your PCR reaction volume (e.g. double everything to go from 50 μl to 100 μl) but you shouldn’t! Instead, you’re better off doing multiple reactions and pooling them together. Don’t worry - it’s not too much extra work. You can still prepare it as if you were doing a 100 μl reaction but then splitting that into 2 tubes.
blog; bit.ly/pcryield
This is because, although the components of the reaction might be able to scale evenly, the heating & fluid dynamics won’t. It will take the liquid in that larger volume longer to heat and cool which will mess with how long your reaction really is at each temperature you want it to be at each step. Although many modern PCR machines have settings where you can put in the volume of your reaction and it will try to account for that, but you still have differences in liquid dynamics and surface area vs volume in different parts of the tube, etc. Bottom line: that PCR reaction you optimized the heck out of at 50 ul might not work well with 100.
Though I must say that, instead of spending tons of time optimizing to get the max out of each reaction, it’s typically easiest to just pool them once you figure out conditions that give you a single nice clean band on the gel, even if that band isn’t as bright as you’d ideally want.
In terms of optimization, parameters you can play with to increase yield include adding additives like DMSO or betaine or formamide which will make it easier for the strands to come apart and loosen up secondary structures so the primers can find the desired binding sites. That can increase your yield. But most of the optimizing you will do is probably to increase your specificity. That’s the most important thing. And you don’t want to mess that up in hopes of increasing your yield.
It also might be tempting to increase your yield by upping your template and/or primer concentrations. But if you do this you risk depleting the other ingredients early on and most importantly you risk nonspecific primer binding and thus more of those nonspecific bands.
Finally, you might be tempted to increase your number of cycles but this risks more typos and their propagation.
Promega has a nice guide on optimizing your PCR: www.promega.com/resources/gui...
And here are some links to other posts of mine
more on PCR: bit.ly/pcrtrain & • PCR (Polymerase Chain ...  
more on nucleic acid spin columns: bit.ly/spincolumns & • Spin column nucleic ac...  
 
more on agarose gel electrophoresis: bit.ly/agarosegelcompare & • Agarose gel electropho...  
 
more practical lab tips: bit.ly/lab_tricks_page  & • Practical lab tips & t...   
  
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@user-ie5sw5xo6m]

Thank you for sharing this. There is so few people on Yb that talks about this stuff❤

[@jyogi30394]

Hi. Can you do a detail video on analysing and interpreting qPCR data, please

[@stefanodecler149]

Nice tip, thanks :) It is necessary to follow de classic miniprep procedure by adding binding buffer and wash buffer? Or it is sufficient to add the PCR reactions to the column, incubate and simply elute out the PCR product? Thanks in advance!