Hi, my name Is Linda and I work in Dr.Edwin Niki's lab at the University of California Irvine. Today I'm gonna show you how I use stigma's cyber green, extracting a tissue PCR kit to rapidly genotype or transgenic mice. Usually I use this kit to genotype our embryonic mice when I'm doing acutely dissociative cell cultures and I need to rapidly, quickly find out the genotype of our mouse.
But for today's purposes, we have adult mice tails in which we can show you how I use this kit. This protocol involves a quick ale digestion and DNA extraction, and then a quick neutralization step. And we use the extract DNA in a real time PCR, where we can monitor our PCR Amplicons.
So if you're Ready, let's get started. So before we get started, We wanna make sure that our heat block is set to 95 degrees Celsius, that our cyber green is thawed and is kept on ice until it's needed and that our other reagents are thawing to room temperature. In my ice bucket, I have my two primers that we'll be using to genotype and they've been specifically designed for our gene of interest and validated for QPCR.
I also have my positive control for the gene of interest and my negative control and a tissue sample that I've already collected from an adult mouse. Now, when you collect your tissue sample, it's very important to be extremely stringent. When we collect embryonic mouse tails, we always rinse our forceps with 70%ethanol prior to and before each tissue collection.
And we always rinse the tail samples in TBS first you have to make a master mix of the tissue preparation and DNA extraction for each tail, you're gonna want 100 microliters of the extraction solution and 25 microliters of the tissue prep solution. I'm gonna use my P 200 and filter pipette tips to measure out a hundred microliters of the extraction solutions and 25 microliters of the tissue prep solution into a new eend rph tube. You're gonna wanna mix your master mix solution by pipetting up and down.
And so you're gonna want to add 125 microliters of your master mix into your of interest, making sure that if it's an adult mouse tail, that the cut end is completely submerged in the master mic solution and you can finger vortex it and you incubate it at room temperature for 10 minutes. During the 10 minute incubation, I'm going to prepare my favorite green master mix, which involves the cider green master mix that they include in the kit milli Q or PCR grade water, and our primers, some PCR grade water, and our forward and reverse primers. And that's it.
Now that the 10 minute incubation is up, we're gonna have to heat and activate the reaction at 95 degrees for three minutes. Our three minute inactivation is up and now we're gonna add neutralization solution that's provided in the kit and we're gonna add a hundred microliters per reaction and you're gonna mix this by vortexing. After you add the neutralization buffer, your DNA samples can be stored at four degrees Celsius until you're ready to use them.
We actually want our genotype right away. So we go immediately into the QPCR, so my tails are neutralized and are ready. My cyber green PCR master mix is ready and I have my PCR tubes set up on a cold block.
All I'm gonna do now is add 16 microliters of my master mix to each well and then add four microliters of the mutualized extract or four microliters of the positive or negative control to each. Well, all I'm doing is adding 16 microliters of a master mix to each of the wealth. Then I will add four microliters of the neutralized extract to the 60 microliters of the master mix in the well for a total of a 20 microliter reaction volume.
And I'll do the same thing for the positive and negative control. Adding four microliters to 16 microliters with a PPR master mix. Once they're all added, you can cap them with an optically clear eight cap strip.
You wanna be careful to not touch the optically clear part of the caps that are over your samples. So I'm using a Kim wipe to push down on the Caps to make sure that they're sealed. So I've gently vortex our PCR and DNA mix and I've spun them down and now we're ready to put them into the QPCR machine.
What we're gonna do is first we're gonna, we load our samples into the Opticon PCR machine and I'm going to make a new plate set up, which our first well will be our sample. Our second well will be our positive control, and our third well is our negative control specify, find that our reaction volume is 20 microliters in total. Next I'm gonna load a protocol that was designed for the person was rapid genotyping.
This protocol involves a three minute denaturing step, a cycle of 15 seconds of denaturing, 15 seconds of primary kneeling, and then 20 seconds of primary extension. Now the primary extension tank can actually be going drop down to about 10 seconds, and this will have a total of only 40 seconds. For each cycle, we have those three steps cycling about 40 times like a normal PCR, and then at the end of that we take a melting curve.
So we have our plate set up and we have our protocol loaded. All we have to do now is run our QPCR and it's starting at the three minute denaturing stuff. From my experience, using the primers that I'm using and using the tail samples and you looking for the genes that I'm looking for, I know that my positives will start to come up around 25 cycles crossing a cycle threshold of significance.
Now, when that crosses is highly dependent upon your tissue sample and what you're looking for and how much of the DNA is present initially. So we can already see that our positive control has already come up and it looks like our sample is also positive. As you see, it's starting to come up a little bit after our positive control.
We're still gonna have to wait it out because it's only at 27 cycles, but hopefully in a couple more cycles, we can be sure that what our sample is is positive and I can then proceed with my experiment. At this point, you can see the negative control just starting to come up, and that's because I took an excess of tail. That helps you be sure that anything that comes up before this excess negative tail is going to be positive.
So at this point, anything that is coming up before the negative, I would consider positive and I would proceed with my experiment. After the PCR is finished cycling, the machine performs a melting curve analysis. This will help you determine the composition and purity of your product.
With this, you can tell if you chose the positive and negative samples correctly. For instance, the positive sample will have a specific sharp melting temperature peak, which corresponds to a specific PCR product, amplified. Any negative samples will have a non-specific broad melting temperature peak corresponding to non-specific DNA that had been amplified during The PCR.
So I've just shown you how we use Sigma's Cyber Green extracting amp to rapidly genotype adult mouse tails. But this kit can be used with various animal tissues, hair, saliva, and I use it to genotype, mouse embryonic tails. We like to use this kit because it's rapid.
I can get my genotype back in an hour, and it's very convenient and simple. So thanks for Watching and good luck.
