Sample Preparation for the Molecular Biological Workflow

Transcription

Sample Preparation for the Molecular Biological Workflow

Hi, Welcome to today’s presentation Sample Preparation for the Molecular Biological Workflow. So think about your current DNA and RNA workflow. How convoluted is it? How much time do you take to do each of your steps? Do you do collection and bulk processing followed by analytical sample processing. Then you usually have to extract your sample for your RNA and DNA. Then you do you isolation to make sure you are getting the pure products that you are looking for. And then you do your prep for analysis; you have to add in whatever markers, primers or whatever materials in order to prepare for your analysis. So a typical molecular biology workflow has different components to it. You have your chemicals, your consumables and your supplies, and any instruments or kits that you need to do your extraction and analysis. And it usually flows from the sample homogenization to sample processing to your sample analysis. And we like to think that Spex can help you throughout your entire molecular biology workflow.

Let’s first look at sample homogenization. Often there is a question “why do you need to homogenize?” or “how much sample do you need to ensure homogeneity?”. There was a study that if you have about a 10% uncertainty for your process, so your process allows you about 10% uncertainty, if your sample pieces were 5 millimeters, you would need 125 grams of sample. Now if you needed a lower uncertainty, let’s say a 1% uncertainty, which is a higher load of accuracy, then you would need almost 12,000 grams of sample. Now if you bring that down to 2 millimeters, about the size of a crayon, then you’re looking for about 8 grams of sample. If you can bring that down smaller to a pencil point, about 1 millimeter, that’s about 1 gram of sample. And if you can get it down to a fine point pen, about 0.5 millimeters, then you need about 0.13 grams of sample. If you can get it down to sub 0.5 millimeters, then you need even less sample to ensure homogeneity.

So, how do you reduce your particle size? It depends on your materials. It also depends on what type of force you need in order to make those materials break apart. There’s compression force, that’s when you have two opposing forces that come down upon the material and they basically crush it between these two opposing forces. Then you have a shearing force, that’s sort of like your scissors where one force shears against the other. Then you have attrition forces, that’s where like two plates rub against each other and you have the material in between, so they are moving in opposite directions of each other. And then you have impact force, where the force actually hits the material and breaks it apart.

If you are using compression force and shearing forces, you are probably using crushers and cutters, and those usually are for particles that are about 100 millimeters or bigger, so those are for really large particles. If you want laboratory or analytical size particles under 100 millimeters into that micron level, then you’re really going to want to be using attrition and impact forces and you will be looking for things like ball mills, impact mills and different types of grinders and mills.

Now it also depends on what type of material you are trying to study. If your material is really hard, if it’s really rigid, then you are going to be looking for a crusher. If you have a soft material, then you’re more than likely going to be looking for some sort of ball mill or impact mill. If your material is really wet, think about different vitamins or gummy candies and things like that, things that are very malleable. Then the crushers are not going to work for you because those are really intended for the harder, more crystalline types of structures like different types of rock and things like that. So if you are going to use it on tissue samples, we actually suggest that you automate your homogenization process and you do use an automated homogenizer, ball mill or some other type of mill. Now the benefit is it is complete grinding, it is a very fast processing. So, if you think about your current, lets say you manually homogenize, sometimes you don’t get a complete homogenization and you can see the picture you see of a ball of material still there. And when you try to process it, if you use an automated system, you can actually cut out multiple steps in your processing and you’ll be able to process it faster. So, we have done some DNA extractions in about half an hour, compared to the hours and days it would take with a manual process, so it really speeds things up.

We are going to take a quick look now at our Spex line of homogenizers, especially our Geno/Grinder and our Genomax. They are appropriate for all types of tissues, from plant tissue, including your cannabis, to animal tissue. They can be used in the lab for organic extractions, like for pesticides, or for molecular biological extractions for DNA and RNA.

Let’s take now a look at our homogenizers (video demonstration)

Now if you have some samples that are temperature sensitive, they have to be kept cold, or maybe they are particularly hard, like teeth and bone, then you are going to want a little bit more vigorous homogenization and you’re going to need a cryogenic process. And for Spex, that means our Freezer/Mills. You can use liquid nitrogen to cool the sample and then a magnetic impeller will then reduce the sample into a very fine powder.

Let’s take a look at our Freezer/Mill (video demonstration)

Once you homogenize your sample, then you are going to go for the processing for that DNA or for that RNA. You are going to more than likely need to purify that genetic material. So, you are going to do some type of digestion, usually with a ProK, you’re going to bind the DNA to a silica column, you are going to pass the through and you are going to wash that column, and the you are going to elute off that cleaned DNA.

What happens when you automate this process, you put it into the homogenizer, and you use it as a mixer or a mixer/mill. What you get is a higher concentration of recovered DNA. So, we did some studies on different types of tissue, beef loin, chicken , mouse heart. And we found in every case, when you used an automated homogenizer you were able to increase your throughput, increase your amount of your DNA concentration that you got out of your sample. And you got a better quality of DNA, you had much better bands in your gel electrophoresis. So, we found you get better quality and better recovery when you are using an automated, versus a manual homogenization and mixing system. We believe our Spex family of homogenizers and our DNAmax kit are the perfect fit together. They are the perfect combination of DNA or RNA extraction, time savings, money savings, and good efficient results.

We like to say that the DNAmax will give you less time at the bench, so you have more time to spend on other activities. You get more samples in less steps, with lower cost and a higher yield. You’re not wasting all your time and your energy doing your processing by automating your processing and using our DNAmax kits. We also give you more, we give you 75 reactions where our competitors give you 50. We give you options to have kits with stabilizer or without stabilizer. And we have kits for plant and animal tissue which are very competitively priced, and all of these DNAmax kits have been optimized for our Spex homogenizer and they have the methods programmed into our DNAmax.

I’m going to take a look now at what you can do with your isolated DNA and RNA. You can look for common bacterial pathogens like Salmonella, E. coli, so these are very common bacterial pathogens, and if you do any type of work with food or consumer products, then you might be concerned about bacterial pathogens. There are also fungi pathogens, thinks like Aspergillus, or black mold, the aflatoxins, the ochratoxins, the penicillin, fusarium, and Claviceps. So, if you work with any of these fungi pathogens, or you are monitoring for any of these fungi pathogens, we actually have some new pathogen kits.

And now we will look at a little bit of our Eco48 by Spex (video demonstration)

Well, I think we have shown you the way to fast track your molecular biology workflow. We really believe that Spex is your partner in your workflow from sample homogenization through sample processing, to sample analysis. And we have shown you the ways in order to speed up your process and make it more efficient. Hopefully we have shown you how to take your process from convoluted to simple, Spex can help you combine different steps in your workflow and make a faster and simpler process.

Thank you