Видео

When cutting an organ, such as the heart, for explant, you want to have a plan in place before cutting. Often, students can just start cutting up the organ creating a slimy goo, instead of a solid chunk of organ. For explant technique, I like to have defined edges to the chunks, as opposed to a liquid mash at the edge. With a defined edge, cells tend to migrate out from the chunk when they exit mitosis. If the cells do not migrate out and adhere to the dish, you will not be successful. Also, with explants, it is better to have a few really good chunks, than a dish covered in mashed up goo.

Thanks. I’m happy to hear you enjoy them.

TAE is an acronym. T = Tris, A = Acetic Acid, E = EDTA. Tris is a strong base. It has as very high pH. The Acetic Acid is a weak acid. It has a low pH. Mix them together and it makes a buffer. A buffer stabilizes the pH, so it cannot go up too much or down too much. EDTA is a chemical that chelates divalent cations such as Mg^+2. Chelate = Binds to them so they cannot bind to anything else. Mg^+2 is a necessary cofactor for DNase, an enzyme that degrades DNA. No Mg^+2 = No DNase activity = Higher concentrations of DNA. Running electricity in water leads to electrolysis of H2O. H2O --> H+ & OH- The H+ ions can interact with the negatively charge DNA molecule and neutralize it. If the DNA is neutralized, it will not be pulled through the gel to the positive terminal. The buffer will bind to the H+ and OH- ions and buffer them, so they will not interact with the DNA.

So nice of you. Thanks

Not exactly sure the name of the filter. It is the amber filter that came with the IO Rodeo Large Blue LED Transilluminator. I believe the blue light is at 470 nm and the amber filters light to about 580 nm.

@@ProfessorDrewCollop Thank you, much appreciated.

Unfortunately, I will not be posting the Absorbance values. Students use this video to record down the % Transmittance and then they are calculated the Absorbances. If I post the Absorbance values, the students will not need to go through this part of the protocol. To calculated the Absorbance (A) from the % Transmittance (%T) use the following formula: A = 2 - log (%T)

Not knowing any other information, there would be no way for me to troubleshoot this. Are you diluting in Trypan Blue for both methods?

For this experiment you need to use 98% purified BSA. We buy the powder, so you can make up whatever concentration you would like. I believe for this experiment I used a 200 ug/mL stock solution of BSA.

I don’t understand your question. Are you asking me what wavelength was used?

There is no specific brand we use. We have use commercial products and made it up from powder. With either version, watch out for particles in the trypan blue. I find they will always appear given enough time. These particles can through off the reading from the Countess.

I am not a microbiologist, so I cannot definitely say it will work on any bacteria. There are alternative protocols that do exist. The purpose of going through each of the steps was to explain the reason we use each of these techniques. The great thing about science, is that it is always evolving. Once I discover a better technique, I switch to it after I have tested it myself. As a result, the way I run protocols in the lab is completely different today than it was years ago. One caveat is if you are working in industry. If you have a protocol approved for production, it cannot be changed with out going through the approval process again. So it is very important to have the best protocol before submission.

How much is what? The Coulter Counter?

If the egg is not fertilized there will be nothing to grow. That is what an edible egg is, an unfertilized one.

@@ProfessorDrewCollop yes that's what I want. Edible eggs. Can you teach one how to grow it. 😇😇😇

This would be a question to ask an egg farmer.

@@ProfessorDrewCollop I know, but I thought scientist now have a way to grow edible eggs that is not fertilized. Guess it's my imagination, I was just so interested in most of your videos. That's why asked if it's possible to have a edible egg in a lab without a hen. But since it's not possible, it's fine. Thank for responding back, I really appreciate.

There is a process to grow edible meat in the lab now, without killing animals. Search for "Clean Meat" or "Cultured Meat" to get more inform. A process to replace the creation of an edible egg is not cost effective. Hens are cheap and not worth the effort.

Concentration is irrelevant to the volume of your solution. The volume is only important if you are trying to determine the amount of the solute in your solution. As an example, you could have 0.5 mL of a 1000 cell/mL sample. It does not matter that it is only 0.5 mL, the concentration will still be 1000 cells/mL. As the concentration is a fraction, it can be used as a conversion factor to determine how many cells are in your 0.5 mL sample. Mathematically, it would be 0.5 mL X 1000 cells/mL. The mL units cancel leaving you with 0.5 x 1000 cells = 500 cells. Therefore you have a 0.5 mL sample, with 500 cells in it, with a concentration of 1000 cells/mL. Hope this helps.

Thank you for the clarification. Indeed was helpful

I would not advise you drink it after the Biuret solution has been added. It is highly basic and contains copper sulfate and potassium sodium tartrate.

The alkaline lysis technique is optimized for purification of plasmid DNA from bacteria. The alkaline environment keeps the DNA denatured. Bacterial DNA is supercoiled, so even when it is denature, the strands remain in close proximity to each other. The genomic DNA is bound to other macromolecules (proteins & lipids) and will precipitate out of solution during neutralization of the alkaline environment. Many of the kits you buy come with a filtration tube. This will filter out precipitated DNA and allow the plasmid DNA to run through the filter and into the lower collection tube after centrifugation.

The Bunsen burner is there to heat the air in the neck of the flask. Hot air rises, so the air in the flask will flow up and out, helping to prevent contaminates from floating down in to your flask. If you don't have one, you might just have some contamination issues, but hopefully you will get many sterile plates.

You might want to check your audio, as there is voice over for 50% of the video. At the half way mark I state that I am just going to be running the samples for larger number of wavelengths and I stop the voice over at that point, as it would be superfluous to continue speaking.

@@ProfessorDrewCollop Yes; indeed. Could you please share the plots of the final output, on x-y axis? Also, in practical terms, what characteristics can it measure in wastewater?

Sorry, I cannot add the final graph to this video, as this is an activity I get my students to perform after watching the video. If you really wanted it, you could graph it yourself. Plot wavelength (nm) as your independent variable (horizonal axis) and Absorbance (no units) as your dependent variable (vertical axis). The Spectrophotometer measures absorbance of different wavelengths of light by a sample. As a Biologist, I do not work with waste water. I suppose you could analyze the turbidity of the water as a function of how many particles are in it. We have some Chemists and Chemical Engineers, within our department, that work with waste water. If you are really interested, I could put you in touch with them. The Spectrophotometer works best for a single known chemical, with a known wavelength of maximum absorbance.

Sorry, but I do not know of one off the top of my head.

Sounds like you are passionate with what you do. That is so very important. Are you referring to the manipulation of the sex of the chicks? Gender is a social construct. Sounds like a very interesting project. What type of chemicals modify the sex of the chicks. They only gestate for 3 weeks. You must need to inject the eggs very early. How to you ensure sterility. I am unsure as to what kind of guidance you are looking for. I do not take PhD students, if that is what you are looking for. Happy to answer any more questions you might have.

I am sure there must be ample resources to explain the reaction if you were to Google it. In short, the heat usually needed to open up the carbon ring in the sugars. In Bial's test, the sugar is first dehydrated (loses water molecules) to create a new compound. Pentoses create furfural, while hexoses create hydroxyfurfural. These products will react with Bial's reagent, turns it from a yellow solution to a coloured solution. Furfural will turn blue-green. Hydroxyfurfural will turn brown. The chemicals in Bial's reagent include orcinol, ferric chloride and hydrochloric acid.

If you are concerned about RNA contamination, you can treat the sample with RNase.

Astute observation.

This experiment is performed to make cell lines. You need fresh tissue that is not contaminated. Adult tissue does not work great, as the cells are bound tightly together and the cells are not in a state of constant cell division, like in the developing embryo. You will then need to disaggregate the tissue into individual cells, if you want to make a cell line. Without a circulatory system, the solid tissue will die as a result of a lack of oxygen and other nutrients. If you want to stimulate the muscle to contract, that is a completely different experiment. You need to harvest the muscle and find the nerve that stimulates it. Then you can apply an electrical current to activate the muscle fibers. I believe I performed an experiment like this on frogs back in third year Physiology. That was a course that changed my life.

I have no experience attempting to extract bone marrow from a chicken embryo. Sorry, I cannot help you on this one.

I assume you are asking about the age of the embryo. Once a chicken egg is fertilized the embryo will hatch about 3 weeks later. The embryos we used for primary culture in the lab are 2-weeks past fertilization. The embryo in the videos is slightly more than 2-weeks old by a day or two.

You could used different types of yogurt to in an attempt to see the relative levels of protein in them.

I believe you emailed me this question already. If not, everything is autoclaved when we are done with it. In terms of references, I don't really have one. The protocols tend to evolve semester to semester as I learn new things or experiments don't work.

Are you looking for the protocol on how I load the hemocytometer, or are you interesting in my gating protocol for BHK cells?

Your gating protocol please. I have started setting my own gating protocol and have been comparing results to our manual counting method. I have no other means for comparison, and I contacted ThermoFisher but they didn't provide much support. Thank you. @@ProfessorDrewCollop

I had the same issue with ThermoFisher. I contacted them to get their gating protocol, but they offered no support. I really don't understand why they could not hired someone to go through the most popular cell lines and create protocols. Here is the protocol I am using for BHK. Not sure if it is perfect, but it works for my needs. I optimized it using a Coulter Counter and a manual hemocytometer to compare. Within the Countess appropriate range, it seems give approximately the same count numbers.

Sorry, the image would not add so here it is typed out. Count setting both checked for Auto FL Threshold and Auto Lighting Size Gating = 12 - 28 Contrast Gating = 0 - 255 Roundness Gating = 0 - 75 Let me know how it works for you. Good luck.

Thank you very much @@ProfessorDrewCollop I'll try it out asap. Many thanks!

Happy to hear you found them useful.

@@ProfessorDrewCollop how many days it has the embryo ?

I was not familiar with Fehling's reagent. I looked it up and do appear to be similar. Fehling's reagent contains a strong base; whereas, Barfoed's contains acetic acid. From what I read, with Barfoed's test, different carbohydrates react at different rates. As an example monosaccharides react quickly but disaccharides react more slowly. As a result you can more easily distinguish mono- vs di- saccharides. What has been your experience with Fehling's reagent?

The majority of preclinical testing is performed on cell lines. To create a cell line students learn to extract tissue and disaggregate (break it down into individual cells). In addition, if you have a cancer diagnosis, you might have a biopsy taken to analysis the disease. Cell lines can be made from your cancer tissue and oncologists can test out different chemotherapeutic drugs on the cells in the lab. This way they can eliminate the drugs that will not work on your cells and they can focus on the best treatment for you before the cancer has a chance to spread. I hope this helps explain the importance of learning this technique.

Are you asking me to show you an image of a successful cell line?