Welcome to the iQ Check
easy extraction deepwell protocol
Here I am in the lab. This is the DNA-negative area;
and the DNA-positive area.
I've got my handy quick guide
so I don't forget what to do next.
Let's get started.
First, we're going to pipet 100 microliters
of the lysis reagent, which is on the stirrer.
It has a stir bar inside to keep everything homogenized.
Now, if you are working with something like Listeria
for example, and you need grinding beads
you have to add those at this point.
So let's add 100 microliters
keep the bottle on the stirrer
and for the sake of time, we're just going to add it to a couple of wells.
Now we're ready to add our sample.
You'll notice I only have one in here
but imagine this tray is completely full
I'm going to open up the stomacher bag;
these samples have been at 37 degrees for about 21 hours
I'm now going to pipet out 100 microliters from each sample
kind of smoosh the bag to get the solution up to the top so the pipettor doesn't go in
- just the tip.
we're going to add that in.
And le'ts imagine I have a second sample here;
take out 100
and add that into the next well.
OK; now we've added lysis reagent and the sample.
The next thing to do is to cover.
So let's start out;
put on the plate cover,
and you can use a shiny side to smooth it down.
Make sure there's no air bubbles.
Now you pop it onto the agitator
you want to go for...
let me check the handy protocol - we're going to do
95 to 100 degrees for 10 to 15 minutes at about 1,300 RPM.
Let's get this going.
Now we can go over to the DNA-negative area and create our PCR mix.
Here we are in the DNA-negative area.
I've already changed my gloves, and I've made up my PCR master mix
with my probe and amplification mix
using the table on the back of the Quick Guide.
I've remembered to include the positive and negative controls as well.
So now I'm ready add 45 microliters of our PCR mix to the plate.
At this point, you want to be careful not to introduce any air.
You can check in at the pipet tip as you as you pull up the solution to make sure
there's no air as you add it to the PCR plate
There we go - 3 added.
Now we're going to take our positive and negative controls
along with our PCR mix plate
over to our DNA-positive area.
OK; let's put our plate down
and our positive and negative controls into the block.
Grab our samples
We want to let this sit for 15 minutes on the bench
or 10 minutes in the fridge
Let's pretend we've already done that.
Now we're ready to transfer our sample into the PCR plate.
Handy multi-channel pipet for this
You're going to want to use
the side of the tip here as a guide
so that you don't push the whole tip into the bottom of the well.
So let's give this a try. You want to transfer 5 microliters
push in the tips; there, you can feel it.
Now you want to just bring up your sample
and pop it in.
The last thing to do is add your positive and negative controls to the plate.
Now you can cover your plate with optical caps.
The plate goes into the machine.
If you have large number of samples you'll want to use the CFX.
and hit "Run" in the software. That's it!
Come back, and you have your results printed out.
You can do some great data analysis, and look for your positives and your negatives.
Thanks for joining us. If you have any tips that you want to share
email us at labnotes@bio-rad.com, and please subscribe to our YouTube Channel.
See you next time!