The first step in our 16S rRNA gene sequencing flow is to extract the DNA.
00:12 - Today we will be looking at two different types of DNA extractions.
00:15 - The first is called the KingFisher extraction, where we set up plates and place it on a machine and the machine uses magnetic feeds to extract the DNA for us.
00:24 - The second type of extraction we will look at today is the QIAGEN AllPrep extraction.
00:30 - This will extract both DNA and RNA and elute them separately.
00:34 - For our extractions, we use a biosafety cabinet to keep all contaminants out.
00:40 - Even though our samples are considered BSL-1, which means they are not harmful to humans, we keep them inside the biosafety cabinet to protect the samples themselves from bacterial contamination.
00:52 - To do this, we use our PPE, which is our protective equipment.
00:56 - So here I have a temp lab coat as well as gloves, taped, and I will place one more pair of gloves over.
01:12 - Our first step is going to be to make the lysine binding solution.
01:16 - This will be placed in the lysing matrix e tube so that we can lyse the cells and release the DNA for extraction.
01:28 - So first we will take the lysine binding solution concentrate and add this to a 50 ml conical.
02:00 - Normally we would add more than this but for the video’s sake and for ease and clarity, I’m just adding a little bit.
02:08 - We then add the carrier RNA and we will then vortex this to mix.
02:32 - Our next step is to prepare the bead mix. These beads are the magnetic beads that will help the KingFisher extract the DNA.
02:41 - We’ll begin with an empty 1. 5 ml sterile tube and we will add a 50:50 ratio of nucleic acid binding beads, as well as a lysis enhancer.
03:10 - Before adding the binding beads, it’s suggested that you invert to mix up the beads.
03:39 - Now that we have our mix, it’s suggested to vortex before putting away in the 4° if you’re not going to use right away.
03:49 - While one person is making the bead mix and the lysine binding solution, the other person can be working on the plates.
03:56 - In total, we will have six plates for this extraction.
03:58 - Five of them will be deep well and the final one will be a tuner μL plate.
04:04 - The first two of the deep well will be wash one solution, the second two will be wash two, and the final plate will be the sample plate.
04:12 - The last plate, which is the tuner microliter plate, will be the Aleutian buffer plate.
04:18 - So to prep all of these plates, we take one deep well at a time, add our wash concentrate -before starting the extraction you should add the isopropanol as suggested- and then multi-channel pipette from the boat into every single well in this 96 well plate.
04:55 - Now it comes to the sample. So the sample will be added to the lysine matrix e tube along with the lysine binding solution. This will then be homogenized and spun down to leave a supernatant which is a layer of liquid above the beads.
05:13 - The supernatant. also known as the lysate, will be transferred into our sample plate.
05:33 - This is the sample itself. Our next step is to add isopropanol and finally the bead mix.
06:20 - Normally we would do these one step at a time for all 96 wells but again, for simplicity and clarity, all three are added to one well.
06:34 - Our final step will be adding all of the plates to the KingFisher machine for the extraction.
06:40 - From here we’re going to load the KingFisher machine.
06:43 - So once you have your protocol loaded onto the machine, it will tell you the order of the plates and when they go on.
06:49 - So our first will be the tip plate. These tips are what attach to the magnetic beads and carry the DNA and RNA through the extraction.
07:00 - Always make sure to line up A1 in the correct corner.
07:07 - Next will be our Aleutian plate, and wash two, and so on.
07:25 - We move on to our wash one, again making sure that A1 lines up in the correct corner, and finally our sample plate.
07:40 - From here, we press start. The run will take about 30 minutes and once it’s complete, you can come back and take your samples that will have both DNA and RNA eluted into the same well.
07:59 - This next extraction is the QIAGEN AllPrep DNA RNA extraction.
08:04 - In this extraction DNA and RNA are eluted separately.
08:07 - So, we have two columns. The first is a pink column, which is an RNA column tube, and the second is a white and purple, which is our DNA column.
08:20 - In a column extraction, the nucleic acid -whether it be RNA or DNA- is caught in the column, and we wash any impurities (such as the opposite nucleic acid or proteins) through the column so that our final elution is pure RNA and DNA.
08:38 - Next we add both our tissue and the Buffer RLT+ into a lysing matrix e tube.
08:45 - My tissue is already in here, so I’m going to add the buffer.
09:04 - Once the sample is centrifuged, we will add the supernatant into the DNA spin column.
09:41 - Once we’ve added the buffer to the spin column, we’re going to centrifuge it down to push the fluids through the column.
09:55 - We will do this after every step. Our next step is to take the DNA spin column (which by now has trapped the DNA) and place them into new collection tubes.
10:14 - We will save the flowthrough for RNA purification.
10:19 - From here we will add ethanol, mix the ethanol and flowthrough, and transfer the mixture into the RNA spin column.
10:58 - First we’ll go through the genomic DNA purification step.
11:03 - With our DNA column, we will add our first wash buffer.
11:25 - We will then spin down this column to produce the flowthrough.
11:30 - From there, we would release the flow through into a trash can and continue with our aw2, or our wash two.
11:42 - We would repeat the same steps: spin it through, discard the flow through.
11:49 - And our final step would be the Aleutian step.
11:52 - In the Aleutian step, we add our buffer EB.
12:21 - We would then spin this down and transfer any flow through to a collection tube as our final DNA product.
12:37 - After the DNA extraction, we need to verify the quantity and quality of the DNA.
12:44 - To do this we use the Nanodrop, which is a spectrophotometer.
12:47 - This uses the absorbance to calculate both the quantity and quality of the DNA.
12:55 - First we will load the DNA samples onto the Nanodrop spectrophotometer.
13:04 - We do so by pipetting two microliters of the DNA directly onto each pedestal.
13:20 - From here we will choose DNA. And from here it will give us the concentration, as well as the 260⁄280, and the 260⁄230 ratio.
13:50 - Based on these numbers, we will be able to determine the quality of the DNA.
13:56 - Once we have ran all of our samples, we can take a look at the data.
14:00 - We would expect our concentrations to vary depending on sample type -in this case we’re looking at fecal samples, and we would expect our concentration to be high.
14:08 - We always want our control to be the lowest concentration, otherwise we might be concerned that there’s contamination.
14:23 - After we have verified the quality and quantity of our DNA using Nanodrop, we will run polymerase chain reaction, or PCR.
14:30 - We use PCR to target a specific gene and amplify -or make many copies of- that gene.
14:35 - In this case, we will be amplifying the 16S rRNA gene.
14:40 - To begin we will decon all of our materials needed and place them into a PCR hood.
14:46 - We use a PCR hood to keep everything that we want in the hood and everything that we would not want -such as contamination- out.
14:53 - To begin PCR we will decon a PCR hood and decon everything that we place into the hood.
14:59 - From here we can turn on a UV light to remove any contamination such as bacteria that may be present still on our materials.
15:10 - Our first step will be to make a master mix with all of the necessary reagents for a PCR reaction.
15:15 - We’ll begin with dNTP mix, PSA, 10x PCR buffer, a forward primer, and TAP.
15:23 - You will then add all of these into your master mix.
15:29 - Next we will begin to load our PCR plate. In each well will be a different reaction, containing the master mix, the reverse primer, and the sample.
15:40 - We will run each sample in triplicate and include a no template control.
15:50 - Now I have my master mix, DNA, and reverse primer combined into each reaction well.
15:56 - I will finish by sealing off the plate with a PCR seal.
16:04 - spinning it down in the centrifuge, and adding it to the thermocycler.
16:12 - Our last step of PCR is to place the PCR plate on the thermocycler.
16:17 - The thermocycler will use heating and cooling cycles to amplify the 16S rRNA gene.
16:33 - Our next step in the 16S workflow is going to be to run a gel electrophoresis.
16:37 - Here we’re going to take our PCR amplicons that we just finished the 16S PCR with and dilute them with water into another plate and from here we will add them onto the gel.
16:52 - The gel will help to separate them out by charge as the DNA travels and we’ll use a ladder to track at what length the base pairs are.
17:04 - First I’ll begin by adding the water to the dilution plate.
17:22 - We always start with water because it is a lower risk of losing over the PCR amplicons.
17:32 - Next I’ll add the amplicons. Next we’ll load our gel. Because the gel has ethidium bromide in it, we need to wear goggles.
17:54 - From here we just gently tear open, being careful not to rip the gel wells.
18:37 - And here you can see the different gels and each of the wells.
18:42 - And what we’ll do here is we’ll load each well with a different sample.
18:46 - We will load both our amplicons and our NTCs -which if you remember is our non-template controls- and from there we will run the gels and as the DNA moves, it will show how far and the length of the band.
19:01 - First we’ll load our PCR amplicons, and then we’ll add our ladder.
19:43 - Next we’ll load the gel. While this isn’t the gel that we ran, this is a really good gel to take a look at.
19:57 - So here we can see the ladder is run on both the right and the left side of the samples, and on the odd wells we have the PCR amplicons, and the even wells have the no-template control, or NTCs.
20:13 - So what we expect to see here is that the odd numbers have nice bright bands, and our NTCs shouldn’t show any bands at all.
20:23 - We expect the bands to occur around the 380 base pairs, and as we can see here, it’s just between the 300 and 400 base pairs, so exactly where we would expect.
20:34 - So these here are 16S rRNA gene PCR amplicons.
20:47 - To recap what we did today, we took our samples and extracted DNA.
20:51 - Following that we quantified the DNA using Nanodrop, and used PCR to amplify the 16S rRNA gene.
20:59 - We verified these amplicons using an e gel.
21:07 - We quantified the amplicons using Qubit. And finally pulled the amplicons, cleaned them up, and sent them off for sequencing.
21:18 - Thanks for joining this section of the QIIME 2 workshop!.