How does contamination affect a PCR reaction?

Introduction

So, we're going to go ahead and add up all our reagents, but I want to make sure that this area is as clean as possible because we don't want any contamination in our PCR machine. If my samples are contaminated with some other substance or if there's something floating around in my water bath then it can really affect the efficiency of my PCR reaction.

So the first thing to understand about contamination is that it's everywhere.

So the first thing to understand about contamination is that it’s everywhere. You can’t get away from it; no matter how hard you try, it will follow you wherever you go. It’s in the air and on your clothes, and if you think about it for a moment, there are probably some bacteria on your skin right now as well. Bacteria are everywhere—in fact, they outnumber human cells in our bodies by ten to one!

OK, let's go ahead and turn that pump on and we're gonna go ahead and start sucking up the liquid into our pipette.

Now that we've got our samples ready and our pipette is all set up, let's go ahead and turn that pump on and we're gonna go ahead and start sucking up the liquid into our pipette. So firstly, you want to make sure that all of your equipment is sterile. So make sure you're using sterile equipment. And also, if your sample has been sitting out for a while, you may want to heat it up a little bit so that it's more fluidic for easier handling. To do this simply flip over your tube or bottle with your sample in it and put it in an incubator at about 90 degrees Celsius for about five minutes or so before starting the experiment.

So now we've got our liquid ready to go into our pipette but before we do anything else with these samples, let's make sure that everything else is clean by checking each piece individually against a UV lamp (which will show any contamination). First off though, check the tip of your pipette: if there are any visible deposits or debris then wipe them away with alcohol wipes until only clean glass remains; then proceed to wash out each section separately under running tap water until no leftover dye comes out when shaken vigorously upside down; finally give each section another quick rinse under tap water just prior to use.

In order to prevent contamination in PCR, you need to take steps to prevent, recognize, and control it.

In order to prevent contamination in PCR, you need to take steps to prevent, recognize, and control it. The most important thing is to use separate equipment for PCR (as opposed to using the same equipment as other experiments you're doing). This means that if something goes wrong with one experiment, it won't affect another.

Use aseptic techniques when handling your samples: keep everything clean and sterile so germs don't spread from sample to sample. Also use disposable pipettes that are only used once in a single tube of liquid so there's no chance of cross-contamination between different samples or tubes of liquids. If possible, place UV light over your work area so that any bugs lurking on surfaces will be exposed by the light before they can contaminate your experiment! Lastly but most importantly—use specific protocols for each type of sample being analyzed because these protocols will be designed specifically for those types of materials; this way there's less room for error!

We need to be sure we won't introduce new contaminants as we're doing our PCRs.

• Be careful of cross-contamination when starting a PCR reaction. Use separate pipettes, tubes, and other equipment that doesn't touch the reagents you're using. Wash your hands before you start the reaction. Use a clean area to work on and make sure that the area is clean before beginning your PCRs.

And over here you can see a whole bunch of different types of contamination that might be in your sample.

Contamination is a common problem in PCR reactions. The environment where you run your PCR makes a big difference, and so does how carefully you prepare samples before running them through the machine. It's also important to note that contamination can come from anywhere: even just one dirty pipette tip can ruin an entire batch of samples! Let's take a look at some examples of contamination sources and their effect on PCR reactions:

Now probably the simplest way to avoid contamination is just to make sure you add up all your reagents in a separate location away from your PCR machine.

The most important step is to make sure that you have a separate room, bench, workspace and set of equipment for your PCR machine. This can be a little inconvenient but it's the easiest way to avoid contamination. It's also important to use a separate set of pipettes when working with your PCR machine so that you don't inadvertently contaminate them. Finally, it's best to use separate tube racks and reagent bottles so that there is no chance of cross-contamination between samples as well as between new and old samples.

So how do we know if there's contamination in our sample?

So how do we know if there's contamination in our sample? You can look for the following:

• The presence of many different colonies that are all resistant to the same antibiotic (such as both ampicillin and kanamycin) growing on your plate. This would indicate contamination with a plasmid containing these genes.
• A pink colour when you add X-gal to your PCR product, indicating the presence of DNA from another organism with its own blue colouring.

One way that people try to control contamination is with ultraviolet light.

One way that people try to control contamination is with ultraviolet light. Ultraviolet light has been shown to be effective against many types of genetic contaminants, such as bacteria and yeast. However, it can also be dangerous if used incorrectly, and most universities or companies will not allow you to use a UV-C light in your lab because they are expensive and difficult to find.

If you have a lot of stuff floating around in your sample, then UV light isn't going to completely remove this stuff from your PCR reaction.

It's important to note that UV light can only remove contaminants from your PCR reaction if they are floating around in the solution. If you have a lot of stuff floating around in your sample, then UV light isn't going to completely remove this stuff from your PCR reaction. However, contamination that is bound to your DNA will not be affected by the UV light at all because it is protected by the DNA backbone. In addition, contaminants bound to reagents like dNTPs (the nucleotides used in PCR) aren't affected by UV light either!

Another way to amplify a DNA sample is through repeated PCR reactions.

If you're not able to use UV light, or if you don't want to use it, one of the simpler ways for people who don't have access to UV light or who don't have time—or maybe they're just not comfortable with using UV light—is just repeating the PCR reaction over time.

So, this is something that I've done many times: I'll set up a master mix and then I'll set up my reactions, such as they are, in tubes. And then when those reactions are finished and amplified, I'll take my tubes out of the PCR machine and move them into another room where there's no contamination. Then we repeat our PCR reaction once again using new primers—typically we'll change out our primers every few days in order to avoid any kind of contamination issues from previous runs on those same primers—and also using new master mix reagents as well as new water; so everything is brand-new each day when we do our PCR reactions.

Conclusion

So hopefully this video has helped you understand how contamination affects your PCR reaction. And if this is something that is important to you, then hopefully it also helps you find ways to avoid contamination in your own laboratory setting.