Video

The images of the slide you saw were filmed at 100x (that's the 10x objective) and at 400x (that's the 40x objective). We didn't film it at 4x objective because it would not have shown as much of the detail that we wanted our students to see, but the "10x" is there. With the 4x objective, you would just have seen more of the field of view, so you would have seen more cells and they would have been about 2.5 times smaller than with the 10x objective.

The part that is not shown in the video (because it's explained in the lab) is that you take the % change in weight and graph it against solution concentration. This will generate a graph with some points above the x-axis and some below the x-axis. You would draw a line of best fit for this data and look for the point at which the line crosses the x-axis (the x-intercept). That is the point (ie. the concentration of sucrose) at which the potato cells would not increase or decrease in size. This means that this concentration of sucrose would be isotonic to the potato cells. Therefore you have just determined "the concentration of stuff inside the cell".

Glad it was helpful!

Thanks

I have no experience with orchids. Please scroll down to some earliest comments to this video - I linked some resources there.

Great to hear!

no

Sorry sir, one more question, what speed does the centrifuge use?

This is E. coli. The strain we use for this is specifically engineered to allow this to happen - this won't work with just any bacteria. In this case, the Bacteria's own beta-galactosidase gene is mutated in such a way that the enzyme subunit that is produced will only work if combined with the beta-galactosidase subunit encoded on the vector.

the centrifugation speed used in this lab isn't that important. It just has to be fast enough to pull things to the bottom of the tube. (I think we used the maximum speed in this case)

Do you mean SDS? No it's not the same.

DNA, on its own, does not fluoresce. While it is true that we use UV light to make it "glow" on a gel, that glowing is not because of the DNA itself, but because of a common additive in agarose gels called ethidium bromide, which binds to DNA while it migrates in the gel.

2-3min should be enough. The purpose of this is just to get the water vapor (remember that you had just heated the sample) to condense on the side of the tube. 2-3min should be enough to time to cool things down.

Thank you sir. One more question, what about the TE buffer volume to dissolve 1 colony bacteria?

The actual volume isn't really that important (I think we used 200ul in the demonstration) - you just need some liquid to resuspended the bacteria. Please note that whatever volume you use, make sure you measure it accurately because you will need to have an equivalent volume in a balance tube for centrifugation.

Best of luck!

@@ItsLearnable Thank you! It is going really well.

That's true, but not all reagents in the lab can be calculated in this way. Some compounds may have some H2O molecules associated with them even in the "pure" form that you receive from the supplier. This affects the formula weight of the compound and will cause your calculations to be inaccurate if you just rely on using the periodic table. It is always a good idea to check the reagent bottles in your lab for the formula weight - just to make sure your calculations are correct before weighing anything out.

Most welcome!

Please keep in mind that this video is meant as a general protocol for making just about any solution - not just a 1M NaCl solution. I this case, I added 50ml of water at the end because the calculations were made for 100ml. So, if I didn't add the 50ml, my concentration would actually be 2x as high as it should be. I ask students to start with about half the volume, when learning to make solutions, because I want them to develop that habit. Sometimes, when making solutions, you need to adjust the pH. In those cases, the act of adding acid or base, will change the volume, so you need to make sure you leave "room" for that. By developing the habit of starting with a lower volume, you avoid the problem of overshooting the total volume in solutions you need to pH.

You're welcome!

Hi, sorry for the late reply. This video goes over the general protocol for making a solution. It doesn't really focus on the calculations that much. I don't currently have a video on calculating normal solutions (partially because I see Molar solutions used far more commonly in a molecular biology lab), but this site explains it fairly well: www.labce.com/spg931723_what_is_a_normal_solution.aspx

Yes.

@@ItsLearnable Thanks! And how much of the supernatant is being added to the master mix?

@@jennaneumann8201in general, it depends on how much or how little target material you think you have in there. In our case, we used 2ul. Since we're testing for the presence of a DNA target cloned into a plasmid, and each cell in the colony has hundreds of copies of that plasmid (we used a high-copy-number plasmid), we will potentially have lots of the target DNA there. So, you could use less than 1ul. The reason I used 2ul is because our p20 micropipettors have a lower limit of 2ul.

We use the full amount (1ml) because of the type of cuvettes we have. There are some cuvettes that have a 0.1cm pathlength, which allow you to use much smaller volumes to measure your DNA or RNA samples, but we don't have those cuvettes in our teaching lab.

using a cuvette with a smaller path-length would also require you to adjust your calculations to account for that. The calculation we used, assumes a path length of 1cm (this is standard for most cuvettes)

@@ItsLearnable Thank you so much 🌹🌹🌹🙏🙏🙏

I use this as a general rule for any enzymatic reaction. This is because the reaction will start the moment the enzyme and substrate are in the same tube (even if the conditions aren't yet what you intended). So, I always tell my students to add the enzyme last. This way you have some control over when the reaction begins, ie. all your tubes can start the reaction at about the same time. In this case, depending on how you plan out your work and on how quickly you work, you could potentially add the Taq polymerase to the master mix. But in our case (this is an introductory molecular biology lab), it was likely that the master mix would be sitting in a tube for some time before being added to the template. So, it was simply better to have students make sure everything else is finished and ready to go before they added the Taq.

@@ItsLearnable thank you very much, That is the best explanation 😍😍😍

Thank you 🙂

@@ItsLearnable the best 😍🧬

@@ItsLearnable glad to find your channel

There is no need to cut the DNA - DNA polymerase doesn't need free ends on the template in order to work. In fact, we were trying to amplify a cloned gene on a plasmid here. Also, in this experiment we were not purifying any DNA - we were using the cells directly.

@@ItsLearnable thank yooooooouuuuu, I am gonna recommend your page in my Instagram.

Which table? The one here: czcams.com/video/Z5hDj2MH60o/video.html

Thanks!

Thanks!

You bet!

Thanks!

Thanks

In the calculation of concentration of the sample, what does 50ul denotes? Vol of what? Thankyou

Thanks for asking the question @@lona6061. It's nice to see people trying to figure out *why* things are done in a particular way. 👍 If you're referring to this: czcams.com/video/O8FytCl_99Y/video.html then please note that it's not a volume. The "50" here is a constant - it's actually the inverse of the extinction coefficient of DNA [0.020 (µg/ml)-1 cm-1 ], and we're using the Beer-Lambert equation to determine the concentration. According to the equation: Concentration = Absorbance / (extinction coefficient * path length). And we have to multiply that by the dilution factor to get the actual original sample concentration. For many students, in what is an introductory course to molecular biology methods, the above might be a bit scary/intimidating. That is why we generally just simplify it, as I showed in the video.

Now it's super clear! Learning and preparing for research watching your videos

I'm not quite sure what you're asking for. This is just a video showing how to make solutions in general.

The purpose of our lab was practicing the skill of preparing simple slides. We were not trying to introduce any plant anatomy or histology here. Since we were aiming at producing a single layer of cells, there wasn't going to be much plant material on the slide. And seeing any detailed structures of the cell interior would be difficult since these cells are not stained with anything (most cell organelles are actually completely colourless). The only thing that has any colour on this slide is the lycopene (that's the red pigment in tomato cells). The only reason you're seeing any other organelles, like the nuclei (czcams.com/video/cI2YjUc42WY/video.html), is that we turned up the contrast using the condenser aperture diaphragm (See here: www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/condensers/). So there is a limit to what you will be able to see, and what colours you will see on a slide (this is why staining slides can be so important).

@@ItsLearnable Thank you very much for so detailed answer!!!

Great to hear!

No.

That's what centering the condenser lens accomplishes. (the light source is generally fixed)

@@ItsLearnable never fix the light source

I have a feeling we're not talking about the same thing

@@ItsLearnable thats not my problem

The media is Murashige-Skoog media (en.wikipedia.org/wiki/Murashige_and_Skoog_medium). The growth regulator in this case was 1mg/L NAA, but it can vary depending on the species. In many cases, the ratio of auxin to cytokinin is close to 1:1.

Some people seem to make a career out of it at home. I do enjoy some of the videos here: czcams.com/users/PlantCellTechnology

Sorry no. In my experience, it can be a bit variable depending on the plant you're using. In general, it tends to be even, or almost even. In our case, we had some success with just 1mg/L NAA

@@ItsLearnable fascinating thanks, yeah I’ve noticed it’s almost even ratios in most plants, whereas the multiplication and transfer media is like 10 to 1 BAP to IBA 😌🌱

A node from a stem is a great way to propagate plants and doesn't require the production of a callus. (it already has a meristem with stem cells - no need to try to generate any). Using leaves can be a bit more tricky.

Hahaha! The Biosafety Cabinet I'm using is meant more for animal cell culture - it's a bit of an "overkill" for plant tissue culture - but it's what I have available in our teaching lab.

@@ItsLearnable it’s so cool! Yeah you can get away with less, My DIY hood is a HEPA filter blowing air inside garbage bags sticky taped together and it works 😅

Great suggestion! I'll have to see how busy this semester is for me, and what opportunities I will have for filming things, but I'll try to keep that in mind.

@@ItsLearnable Thank u, also can u share your Insta link ? I couldn't find your channel page there.

I'm not on Instagram, that's why you couldn't find me. I've been told that I should have a presence there, but I haven't really taken the time to get involved with that platform.

I'm so glad!

My pleasure 😊

I'm glad it was helpful and thanks for your suggestion. I already have a video dealing with calibrating the microscope here: czcams.com/video/QV45EYqpsd0/video.html . You would simply do this at the magnification you wish to use (though it is likely to be a bit more difficult at the oil immersion lens level). In practice, I normally just set up Kohler illumination at using the 10x objective lens and don't make any adjustments at higher magnifications (but your requirements may differ).