Centrifuges - Science topic

Dear Joshua Van Dyke, why not using a bath sonicator instead of the probe one. My Regards

Depends on the FTIR method you are using, I guess you are using a configuration suitable for measuring liquids. First thing I would do is measure the media-bacteria-dye solution spectrum and media-bacteria solution spectrum. First check that it is correct spectra (absorption intensity, presence of relevant peaks...) and then see if there is any characteristic band corresponding to the dye. If there is, you can quantify dye degradation using that peak, if there is not... try processing the sample so you get a nicer spectrum, maybe.

Dear Dr. Ana Lopez,

I feel the selection of an appropriate centrifuge tube size may solve this problem. Depending on the volume of your sample, you may choose the most appropriate tube size to ensure precise and effective separation as it is a critical factor in optimizing the centrifugation process.

Using a tube that is too large for the sample volume can lead to inefficient separation, as the larger surface area may cause the sample to spread thinly, reducing the sedimentation force on each particle. 

Therefore, matching the tube capacity closely with your sample volume is essential for achieving optimal separation efficiency.

Have you tried using 15ml centrifuge tube instead of 50ml tube for a small volume of 5ml? I feel it should work at the same speed of 1500rpm.

Regards,

Malcolm Nobre

Hello Md Moudud Islam

The protocol seems okay. But you need to consider the points provided below which you may have missed.

1. Tissue may not have been fully homogenized.

2. Tissue may not have been immediately processed or frozen after removal from the fish.

3. Tissue may not have been completely disrupted. If centrifugation is done prior to adding chloroform, there should be a white mucus-like pellet. If there is a tan-colored precipitate, this is indicative that not all the cells have been lysed.

4. If a mortar and pestle has been used to powder the tissue, RNA may stick non-specifically to the mortar and pestle. It may be better to use a glass homogenizer and teflon pestle. Add TRIzol to the homogenizer, then add the frozen tissue and homogenize.

5. Homogenizing for too long and too continuously in a small volume (say 1 ml) may cause heating of the sample and this may result in degradation of RNA in the tissue. Please note that the tissue should be cooled during homogenization, and homogenization should be done in on-off cycle.

6. Use 100mg of tissue sample per 1ml of TRIzol.

7. You may collect the upper aqueous phase carefully without disturbing the interphase. You may collect 80% of the upper aqueous phase and leave behind 20% so that you do not contaminate the aqueous phase.

8. You may try two 75% ethanol washing steps instead of a single washing to remove any remaining impurities in the form of water-soluble salts. It may help to improve RNA purity.

9. Instead of MLQW, use TE. If sample is dissolved in water, the ratio may be low due to the acidity of water or the low ion content in the water. The ratio can go up if the sample is dissolved in TE and the spectrophotometer is zeroed with TE.

Hope these suggestions help to increase the yield as well as the purity of RNA.

Best.

I dont know about the bacteria but for the DNA you can use the QIAamp Fast DNA Stool mini kit.

Bhawna Bhawna Follow the protocol for 3 mL. At the time of centrifugation, simply separate the mixture into two 1.5 mL portions in Eppendorf tubes.

Your idea about gravity within a solar system being stronger farther away is very interesting, but it goes against what scientists currently know about gravity. Gravity weakens with distance according to established physics.

While the concept of centrifugal force balancing gravity exists, it doesn't negate gravity entirely. Similarly, weight should increase closer to the center of mass, not decrease.

If you have detailed calculations and want to challenge the current understanding, consider publishing them in a scientific journal for review by other scientists. Just be prepared to show how your model aligns with existing principles.

For genomic DNA, any standard condition, 15-20 min, 5000 g would work, however, good to use at 4 °C if possible. Time can vary based on your sample density.

People prefer static models, without using G!

To separate bacteria living in the media from diatoms and identify the phycosphere composition of diatoms, you need to apply a method that will establish

1. How to collect and prepare the sample

2. Differential centrifugation

3. Filtration and

4. Analysis of Diatoms and Bacteria

If you so desire the method ; Contact Ogelchemicals@gmail.com.

Hello Iris Yoon,

I have some suggestions which you could incorporate in your protocol.

1. Subculture cells before they reach confluence because cells in over-confluent cultures begin to form rosettes with necrotic centers.

2. Use a subcultivation ratio of 1:2 to 1:4.

3. MM1S is a semi-adherent culture. So, you may have to gently scrap the loosely attached cells with the help of a cell scrapper to get all the cells in suspension.

4. In your maintenance protocol, why do you resuspend the cell pellet in PBS? Use complete growth media to resuspend the cell pellet.

5. You should supplement your growth media (RPMI + 10% FBS) with 2mM L-glutamine.

Hope these suggestions work!

Best.

Just wait for the publication of your paper. Be sure that you are author or co - author of the paper with significant contribution.

The Coriolis Force is not a "force", as such, but an illusion created by the rotation of the Earth. At the Poles, where there is no sideways motion of the ground supporting a pendulum, the force appears to be largest because as the Earth turns around you (because of its rotation) there is nothing to hide that rotation relative to you. But at the Equator, you and your pendulum are being dragged around the Earth at over 1000 miles/hour, which twists the support of the pendulum in a way that hides the rotation of the Earth from your view. The "torque" on the top of the pendulum support is largest at the Equator, but since you can't see any rotation, it appears that the "force" is zero.

In reality, watching a pendulum swing back and forth at the Equator, as it moves northward it twists a smidgen toward the right, while as it moves southward it twists a smidgen to the left (as a result of the torque acting on its support), because the effect is in the opposite direction in the northern and southern hemispheres; and although you could see each of the two small twists with a very high magnification, since they exactly cancel each other, you can't detect any deviation from a straight-line motion with ordinary instruments.

I don't know whether it would help, but my website has a page about "Coriolis Effects" with both a discussion and diagrams I created for my astronomy students, and links to two online videos. Unfortunately, since I wrote the page over 20 years ago, the top one, though it still goes to the linked page, doesn't go directly to the video, so you have to click on the .mpeg link to download the short playground demo (I'll fix that ASAP, but probably not before you read this). However, the link to the outstanding film "Frames of Reference" still goes directly to the Internet Archive's copy. The text I wrote is probably a more useful discussion of the pendulum's demonstration of the Coriolis Effect, but the 1950's movie is well worth watching, even though it only deals with the Effect near the end. (The URL for the page I wrote is https://cseligman.com/text/planets/coriolis.htm )

You could try reducing the concentration of chloramphenicol down to 5 µg/mL, since L. lactis is pretty sensitive to it. If that doesn't work, I've had good luck with NZ9000 using the lithium acetate-DTT method:

The 10^10 CFU/mL concentration of L. lactis NZ9000 for the final cell density they mention is approximately an OD600 of 85 (calculated, from a 1:100 dilution). I can't remember if they mention it but you can definitely freeze aliquots instead of making the cells fresh every time. Also you can omit the antibiotics in the recovery broth, that part of the protocol never really made sense to me.

Is it a protocol from kit's instruction?

If yes, maybe need change incubation times or centrifugation xg. As I know, centrifuging a lysate at 500 xg for 10 min separate nuclei components from others. Followed by centrifugation of supernatant (from previous step) at 10000 xg for 20 min separate mitocondrial components, and centrifugation of supernatant (from previous step) at 100000 xg for 1 h separate microsomes so that supernatant is contained cytoplasmic proteins.

The use of gradient sucrose buffer separate proteins based on their sedimentation coefficient. Therefore you can choose the relevant fraction.

Generally, you must do this protocol and evaluate the results. If need you can change factors to optimization

Just as importantly as them being clean and / or sterile is them still being in good condition. Centrifugation, especially at speeds closer to the rcf limits of the centrifuge bottles, puts a great deal of stress on them. If you are going to reuse them, ensure they are not deformed, cracked, discolored / cloudy, or show other kinds of stress.

Please share any research or review article supporting the answer When synthesizing nanoparticles, what is the reason for centrifugation using ethanol

Try using glycogen to precipitate rna better

Hi Federica,

Freezing likely lysed most of the cells, so centrifugation would make little to no difference.

Hi Kishan,

I think you may be losing cells by collecting them and then spinning them down. I first rinse with PBS one time, then I collect the cells by directly adding Trizol (500ul for a 6cm dish or 6 well plate) to the plate. I do not scrape my cells and I just pipette the solution up and down about 5-10 times to help the extraction. With this method I am able to extract RNA from very few human macrophages. I also do use 1ul of glycogen to help pellet my RNA before precipitation, it also prevents me from accidentally disrupting my RNA if I have a visible pellet (alot of times the pellet is too small to see). I am including a reference for the protocol that I use that was published by former colleagues of mine. Please let me know if you have more questions. Hope this helps.

Skylar

I use 1ul of glycogen to pellet my RNA.

Malcolm Nobre

any tips for the sub-cultivation step?? :)

Generally, nanoparticles do not settle down with 4000 rpm. You can use 10000 rpm or more to separate the nanoparticles.

Priyanshu Srivastava I have thawed a new vial of cells. This time I centrifuged them for 2 minutes at 1000rpm during thawing and my cell pellet looks white and normal. When I sub-cultured them they looked grey and turned black during the second culturing. Is there any problem with freezing or the incubator? I have attached the microscopic image of the cells that I took with my phone.

Hey there Md. Zaved Hossain Khan! So, you're diving into nanoparticle synthesis? That's cool! Let's get started. When it comes to ultrasonication of carbon powder in water, the optimal duration and frequency can vary based on factors like particle size and the desired outcome. Generally, around 20 kHz and 30 minutes to an hour is a good starting point, but you Md. Zaved Hossain Khan might need to adjust based on your specific setup. Now, about using the nanoparticle precipitate without drying... it's a bit tricky. Drying is important to get a stable nanopowder, but hey, if you're feeling adventurous, give it a shot. It might work for your application. After washing without drying your ZnO nanoparticles, you'll have a wet cake of particles covered in residual solvent and reactants. It's not ideal, but depending on your application, you Md. Zaved Hossain Khan could experiment with it. Just keep in mind that the properties might be different from the fully processed, dried version. In the world of nanoparticles, it's like cooking - sometimes you Md. Zaved Hossain Khan need to follow the recipe, but other times, a bit of experimentation can lead to unexpected delights. Good luck with your research!

Hello Mnor Faleh

Q1. When we revive the cells with the flask size, it is better to use a 75 cm2 culture flask or a T- 25 cm2 culture flask?

Whenever you revive the frozen cells use a smaller size flask such as T-25 flask because thawing is a stressful process and there are chances that the cell viability may reduce during this process. So, by using a smaller surface area, the cells would be able to be near each other and communicate with one another in a much better way resulting in speedy recovery from the thawing process.

Q2. Some protocol says when you want to revive the cell, transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium 10% EMEM media, and spin at approximately 125 x g for 5 to 7 minutes, then discard the supernatant and resuspend cell pellet with the recommended complete medium, then dispense into a 75 cm2 culture flask contains 15- 20 ml of 10 % of EMEM the complete growth medium, while other protocol says take the entire content of the vial and aliquot into T-25 flask without centrifugation step? Is it butter to centrifuge or not?

Yes, for less sturdy cells which may not be able to withstand centrifugation, you may place the entire content of the vial into T-25 flask without the centrifugation step. The next day, when the cells have attached to the substratum, you may change the growth media to get rid of DMSO present in the freezing medium. However, for HepG2 cells, you may follow the protocol namely, transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium 10% EMEM media, and spin at approximately 125 x g for 5 to 7 minutes, then discard the supernatant and resuspend cell pellet _ _ _ _ _etc., except for the last step namely, dispense into a T-25 cm^2 culture flask containing 10 ml complete growth medium (EMEM + 10% FBS).

Usually for HepG2, the sub cultivation ratio of 1:4 to 1:6 is recommended. For your requirement as per your question use 1:5 ratio.

Use 5% DMSO.

Q4. Some protocols say 90% of FCS and other says 5% with complete culture media, so which is better FCS or complete culture media? If it is better to use complete culture media, should the media be supported with 10% FBS?

Just use complete growth media which contains 10% FBS and supplement it with 5% DMSO.

Yes, whenever you revive the cells, use 20% FBS in growth media for at least 2 passages after thawing so that the cells fully recover from the freezing and thawing stressful conditions. Return to 10% FBS for the subsequent passages.

Good Luck!

I think it was a good idea to set aside a small portion of the lysate for subsequent quantitation and preparation for electrophoresis if you were not able to do those steps immediately. I also agree with storing the bulk of the lysate at -80^oC if you can't immediately proceed with purification steps. However, it would be better to prepare the lysate when you have time to go directly to the first purification step, since the lysate is the point at which proteolysis is most likely to be a problem. Freezing should be done as rapidly as possible, using a dry ice/ethanol bath or liquid nitrogen.

The concentration should not change during storage, unless there is significant precipitation, so it should not be necessary to repeat the Bradford assay. Make sure the sample is well-mixed after thawing, because some separation of solutes can occur during freezing.

The incubation period for the Bradford is 5-10 minutes. If you wait too long, the protein will precipitate due to the acidity of the reagent.

Vatamanu Sergiu-Marian Hi, I am trying to do zymography and use a lysis buffer with Tris, NaCl, and NP-40, but the sample stays very viscous making it very difficult to load on the gel. How should I process it to use so that the enzyme activity is also retained? Do you have any suggestions?

Thank you

Samir Ranjan Panda

thank you very much for your help

I repeated the experiment with 8 hrs treatment time , and it also seems that the untreated group has higher ROS levels

do you have any further suggestions please ?

Thank you Dr.

If you have not done it, the first thing you need to do is work with known samples. We never test a new technique on valuable samples. Once you are familiar and comfortable with the new technique, and know you can reliably get data, then go on to process the precious samples.

It seems you are comfortable with the SP3 protocol, so it makes sense the problem was earlier on in the process. All I can suggest is what you probably already know, go back and try again with "test" samples.

I have not used the Covaris reagents, but we routinely process FFPE samples and deparaffinisation is a standard protocol that works well in our hands. The only thing to watch out for is that all the wax is removed. Some tissue sections have a huge amount of wax and if it's not all removed it does cause problems further down the line.

Good luck

Hi Feng, I met the same problem recently, have you solved it yet?

Hi Dr. Rebecca

I agree with Dr. Alex about her ideas and in addition to that you may have to try thawing your cell samples gradually as it is done for sperm and ova cryopreservation thawing with liquid N2 -196. Be careful of any moisture or water drops.

Good luck

Dear Dr. Faiz Ahmed Raza

I am sending you the manual containing the troubleshooting for your ROTINA 380 R (Hettich GmbH ) centrifuge machine.

I hope it helps you to solve your problem.

Best regard for you

How much sample do you want to centrifuge and wash? How many centrifuge tubes are you using for your sample?

Glad you found the cause.

Lyophilization of the silver doped quantum dots can be performed. First you need to freeze them using liquid nitrogen and further lyophilize it for atleast 24 hours.

Thanks for the update. Under optimum conditions ("perfect" amplification efficiency), the Ct should shift by about 3.3 cycles per 10-fold dilution (=log₂(10)), hence about 6.6 cycles per 100-fold dilution. The shift will be larger under suboptimal conditions). If your normal samples have Ct-calues of 24 or larger, then it should not happen that 100-fold dilutions of these samples have Ct-values of less than 30 (you mentioned 27). This is strange.

Apart from this I don't see anything obvious that would possibly explain your findings. Does this happen only for a specific primer/assay or do you see this also for other primers? If yes, it might be due to the instrument. If only a particular set of primers/assay doesn't work well you might just design and order (sligtly) different primers, what might solve your problem.

I use the RNeasy kit but for my disruption/homogenization (I am working with zebrafish brains that have been stored in RNALater previously) I just use a pestle and so far has been working well. 1% agarose gel (not denatured) has also sufficed. when preparing my samples for the gel, I make a known amount (ie 2 ug) of the sample and then add DEPC treated water to it. if that volume is 10 uL, my loading dye would also be 10 uL and load 20 uL in total.

The equator is moving quickly as the earth's spins, so it has a lot of centrifugal force. In contrast, the poles are not spinning at all, so they have zero centrifugal force. Since centrifugal force points outwards from the center of rotation, it tends to cancel out a little bit of earth's gravity. Since the centrifugal force depends on →R, it is greatest at the equator and zero at the poles. As a result of the centrifugal force, the Earth has become slightly oblate, with an equatorial radius of 6378.1 km that is 0.34% greater than the polar radius of 6356.8 km. Centripetal force Fe=mv2R and the radius of the earth near the poles are less so the centripetal force is maximum near the poles. The centrifugal force for the spinning of earth is maximum at the equator and vanishes at the poles. Thus, the gravitational acceleration (g) is minimum at the equator and it is maximum at the poles. Specifically, Earth rotates faster at the Equator than it does at the poles. Earth is wider at the Equator, so to make a rotation in one 24-hour period, equatorial regions race nearly 1,600 kilometers (1,000 miles) per hour. Near the poles, Earth rotates at a sluggish 0.00008 kilometers (0.00005 miles) per hour. The earth spins at constant rate but rate of movement is different the equator is moving fastest and poles are not moving (ignoring the fact that earth is orbiting the sun). Because of this movement centrifugal force is pulling matter closer to equator which structure outwards giving earth slightly non-spherical shape. At the equator, the circumference of the Earth is 40,070 kilometers, and the day is 24 hours long so the speed is 1670 kilometers/hour (1037 miles/hr). Thus as the spinning speed of earth increases, then g at equator decreases and thus weight of body decreases at equator. An Earth spinning in the opposite direction would have very different atmospheric and ocean currents. Although the global mean temperature would remain almost the same, the major ocean currents would switch from the Atlantic to the Pacific, changing the planet's climate drastically. The product of mass and acceleration due to gravity is the body's weight, and the acceleration due to gravity increases with latitude. The equator has the shortest latitude and the poles have the longest. As a result, gravity-induced acceleration and weight are greatest near the poles and lowest at the equator. Continental Deflections When surface currents meet continents; the currents deflect, or change direction. Currents are also affected by the temperature of the water in which they form. The major wind belts push the water in the surface currents. The water moves in the direction of trade winds: east to west between the Equator and 30°N and 30°S. When surface currents meet continents, they change their direction. Winds, water density, and tides all drive ocean currents. Coastal and sea floor features influence their location, direction, and speed. Earth's rotation results in the Coriolis Effect which also influences ocean currents. Three forces cause the circulation of a gyre: global wind patterns, Earth's rotation, and Earth's landmasses. Wind drags on the ocean surface, causing water to move in the direction the wind is blowing. Earth's rotation deflects, or changes the direction of, these wind-driven currents. The continents act as barriers to surface currents. When a surface current flows against a continent, the current is deflected and divided.

No, I expanded the cells 1:3.

ok this is compounds that are well known to be volatile.

1) make very sure You are never condensing to dryness. - try always to keep at least 0.5 mL solvent.

2) MTBE and sulfuric acid does not sound like the best idea (as sulfuric acid will protonate the MTBE and transfer this into the aqueous phase.

3) after sulfuric acid treatment I expect You do a small column separation probably on silica gel. - the right activation of the silica (heating, water content) will be critical as well as the eluting solvent system. depending on the activation n-hexane would be sufficient or mixes of hexan/MTBE are needed.

4) if in doubt test each step alone...

good luck. - You are not the first one in trouble

Centrifuges are widely used in forensic science. In Biology Division it is used for separating blood components from blood samples.

Re-suspension: Try re-suspending the pellet in a smaller volume of PBS or a different buffer that is compatible with your downstream purification method. Gentle stirring or agitation might help in breaking up the pellet. Also, ensure that the PBS is at the correct pH for your protein of interest.

Buffer Adjustment: If PBS is not effective, consider adjusting the pH or ionic strength of your buffer to enhance solubility. Sometimes, small changes in buffer conditions can make a significant difference in dissolving the pellet.

Protease Inhibitors: Add protease inhibitors to your buffer to prevent protein degradation during the dissolution process. Protease contamination can cause cloudiness in your solution.

Detergents: In cases of hydrophobic proteins, adding a mild detergent like Triton X-100 or NP-40 to your buffer can help solubilize the proteins. Be cautious with the detergent concentration, as excessive detergent can interfere with downstream applications.

Extended Centrifugation: If you're unable to clarify the solution through a 0.22 um membrane, try a longer and higher-speed centrifugation step. It's possible that some precipitates or contaminants are still present.

Filtration: If centrifugation doesn't clarify the solution, try using a lower pore size filter, such as a 0.45 um membrane filter, before attempting the 0.22 um filter. This may help remove larger particulate matter.

Alternate Precipitation: Consider using an alternative protein precipitation method like acetone or trichloroacetic acid (TCA) precipitation, which can sometimes result in a cleaner protein pellet.

The first step should be adding tpp dropwise into chitosan solution under stirring. The formation of pellet requires high speed, therefore you can try first to check via microcentrifuge tubes at 14000rpm

Thou Chen Loh :)Thank you very much for your reply. I ran the agarose gel test directly and I didn't heat it to 70 degrees before electrophoresis. Do I need to mix the RNA and lodding buffer and heat it to 70 degrees before testing?

Sample prep solution for 2D-PAGE experiments called 2D rehydration buffer which includes non-ionic detergents and urea may work. Recipe like;

(8 M urea, 4% CHAPS, 40 mMDTT)...You may also modify RIPA by increasing the SDS, adding a reducing agent, and adding urea as chaotropic agent up to 8M to induce solubilization...You may also investigate novel methods for increased solubilization at inclusion bodies...

here is one of them;

https://www.thermofisher.com/order/catalog/product/78115?gclid=Cj0KCQjwrfymBhCTARIsADXTabmpmgARsfk_UEnrzRvAvjpRGkKZ2y3Er3Wnqn4PTAXuMAaDtbzLxiYaAuQmEALw_wcB&ef_id=Cj0KCQjwrfymBhCTARIsADXTabmpmgARsfk_UEnrzRvAvjpRGkKZ2y3Er3Wnqn4PTAXuMAaDtbzLxiYaAuQmEALw_wcB:G:s&s_kwcid=AL!3652!3!458037844736!!!g!!!6617619314!79549275115&cid=bid_pca_ppf_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con

Dear Ekaterina Anatolyevna Lapchenko,

Unfortunately, I do not have any experience or knowledge about this subject.

Best regards.

Hi Melissa Stofberg can you explain more on the incubation of BSA? Thank you!

There is actually a formula to calculate this using the k factor method. The k-factor is a measure of the sedimentation efficiency, which is inversely proportional to the time required for a particle to reach equilibrium in a centrifugal field. The smaller the k-factor, the faster the pelleting efficiency: https://lab.plygenind.com/compensate-lower-centrifuge-speed-increasing-time

These plates and plaques look much better. It does not look like the water or condensation is affecting anything, but if you are worried then be sure the plates are sufficiently dry before using. You could leave them for a day at room temp or a few hours in the incubator before you use them.

Thank you Praveen for your answer. I did try several way to ensure the solution go through the PDMS mould using centrifuge but all went south. However, vacuum oven greatly helps in ensuring the solution to really go into each of the PDMS negative mould and creating robust MNs film with less bubble formation.

some plastics are autoclavable and should not release significant chemicals, and you could even leave the cap on during autoclave, just loose the cap to let air in/out, the salt you saw may be due to some evaporation during autoclave: https://lab.plygenind.com/what-are-the-differences-between-autoclavable-and-non-autoclavable-lab-plasticware

Hi Yu Zhang

I briefly wanted to reach out concerning your inquiry. Firstly, Jurkat T cells clump a lot (especially when re-stimulated). To that end I would suggest to spin them and gently resuspend them by pipetting up and down to get a single cell suspension.

Follow the extracellular staining protocol outlined here:

Please stain for live/ dead cells (in PBS) and then for your extracellular antibodies in FACS buffer (i.e. PBS + 0.5% BSA + 0.1 % NaAzide (optional for storage).

For a very basic protocol please refer to:

I hope that helps.

All the best & good luck with your experiments,

Michael

You may try filtration instead: https://lab.plygenind.com/centrifugation-vs-filtration-whats-the-difference-and-how-to-choose-for-your-separation-process

Hi, in our iPSC lab, we have several methods for removing differentiated cells:

I hope this helps!

Best,

Veronika

Used RIPA, 40 Hz, 4,800 rpm, for 1-min pulses, three times, with a 30-s rest on ice between pulses, using carbide or zirconia beads...

Stam Nicolis. "Gravity is related to any force", which is wrong.

Hello Sara,

One possible approach could be:

1-Low speed centrifugation (500xg) for 5-10 min to pellet down the human cells while bacteria will primarily remain in suspension.

2-Take the supernantant to another tube and centrifuge for 10 min at 5,000 x g to pellet down bacteria.

3-Remove supernatant and resuspend the bacterial pellet in appropriate volume to start your DNA prep.

Good luck!

Alex

Only 2 years late.

One of my lab coworker gave me Trypsin powder from the late 80's, it was well conserved at -20°c since then, in its original packaging, itself in a jam pot vacuum sealed.

I believe 3 years in an eppendorf shouldn't be that big of an issue.

The 1st thing I would worry about is whether the homogenization technique is sufficient to break the cell membranes down to the level of small vesicles, because that it what you are relying on during the subsequent differential centrifugation steps. If the membrane particles are too large, they will be in the pellet rather than the supernatant during one or more of the pre-100,000 g centrifugation steps.

Try using a high-power microscope to look for cell fragments after homogenization. You should not see any significant amount.

I found this solver : -

Sometimes cells don't pellet well if there is DNA released from dead cells. What do they look like when they don't pellet? A wispy looking aggregate is often a sign of a DNA problem.

If this is the case you could try a DNAse treatment. You could also try something to reduce cell death like scraping more gently. Or instead of scraping after Relesr treatment, gently wash the cells off the plate with media.

Of course this assumes it is not something basic like the centrifuge note working properly or being set to RPM instead of g (seen this happen quite a few times).

Hi Suh Hee Cook

maybe a stupid comment, but at least in our lab, I would ask. Have you checked that not somebody has changed the setting from g to RPM? But I'm sure you have checked it and other cells you are working with are just forming nice pellets.

Best wishes

Sönke

300g for 10mins should be enough to pellet your cells. I prefer to use eppendorfs or 5ml FACS tubes for smaller volumes as I can see the pellets more easily.

I also recommend aspirating with a pipette tip rather than vacuum until you find out where you are losing your cells.

For anyone interested, the issue seemed to be overall RNA quantity in the samples, which is reduced to begin with in fruit flesh and degrades over time for fruit coming out of storage. We doubled the amount of sample for the extractions and results generally improved. RNA levels also seem to very by apple cultivar.

I think that the cell pellet should be ok without the lysis buffer if stored at -80. Alternatively, if you want them pre-lysed, you can go one step further and add your 4x and the reducing agent to your clarified lysate before freezing. This is how we always stored our samples. It's significantly more stable than storing them in straight lysis buffer and we never had issues with protein degradation even stored at -20.

Yes, there are a few studies that have investigated the effects of centrifugation on RALA peptide vector complexes. In general, these studies have found that centrifugation can lead to aggregation of the complexes, which can result in larger particle sizes. This is likely because the centrifugal force causes the complexes to collide with each other, which can damage the complexes and cause them to aggregate.

One study, published in the journal "Bioconjugate Chemistry" in 2012, found that centrifugation at 10,000 RPM resulted in a significant increase in the particle size of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were less effective at delivering the plasmid DNA to cells.

Another study, published in the journal "Molecular Pharmaceutics" in 2013, found that centrifugation at 10,000 RPM resulted in a decrease in the transfection efficiency of RALA peptide vector complexes. The study also found that the complexes that were centrifuged were more likely to aggregate.

These studies suggest that centrifugation can have negative effects on the properties of RALA peptide vector complexes. Therefore, it is generally recommended to avoid centrifuging these complexes unless absolutely necessary.

If you do need to centrifuge RALA peptide vector complexes, it is important to use a low centrifugation speed (e.g., 5,000 RPM) and a short centrifugation time (e.g., 5 minutes). You should also avoid resuspending the pellet after centrifugation.

It is also important to note that the effects of centrifugation on RALA peptide vector complexes may vary depending on the specific protocol that is used. Therefore, it is important to experiment with different centrifugation conditions to determine the optimal conditions for your specific application.

@ Fatima, it depends. If you properly operated and maintained then to my experience centrifuge can last 10 years or more and CO2 incubator can last 13 years or more.

My guess is either the sonication did not work at all and the cells were not broken, or the excessive amount of sonication overheated the sample so much that all the protein precipitated.

In future attempts, you should monitor the progress of the cell breakage after each cycle of sonication. One way to do this is to look at the cells under the microscope to see if they are intact. Another way is to measure the protein concentration in the supernatant.

in cylinder mesh number of elements increases the number of node

Try soaking the pellet (or region of where the pellet would be) in ice cold 80% ethanol. RNA pellets that come from precipitations using isopropanol tend to be clear. Soaking will allow the pellet to become white. Just note, when precipitating with isopropanol, your pellet will contain more salt. You will need to wash more.

Without details of the protocol you performed, it is difficult to troubleshoot what exactly happened. But try my suggestion above to see if any RNA pellet appears.

Did you observe the same effect in non-cultured conditions?

I mean do you able to dissolve the protein-based sample in pH approx 7.2-7.4 buffer?

Can you also confirm that precipitate and pH change is not caused due to cell culture contaminants (yeast, bacteria, mold, virus etc..)?

pI 4.5 must be still solubilized in pH 7 roughly if other considerations, such as heat-induced aggregation, conformational changes, oligomerization, denaturation/unfolding, or chemical modification, etc...

Is your sample pure protein or some vegetal components have also been accompanying?

Test the gradually decreased pHs alone with your POI to see only the pH effect first, then we may diagnose the problem and propose solutions...

If you suspect that DNA is causing your synovial fluid samples to become jelly-like, there are a few steps you can take to address this issue:

It's worth noting that the viscosity of synovial fluid can vary between individuals and under different pathological conditions. Therefore, it is important to compare your samples with appropriate controls and consider the normal range of viscosity for synovial fluid during interpretation.

By implementing these strategies, you should be able to reduce the viscosity caused by DNA and improve the process of analyzing proteases in synovial fluid samples.

These video playlists might be helpful to you:

Thank you Taufeeque Ali. I will definitly follow your instructions.

Hello Chris Ng

You may incorporate the following steps in your protocol.

Step 1. Washed the cell suspension with PBS three times for 1 mL.

Wash the cell suspension with ice-cold PBS only once to minimize RNA degradation.

Step 3. Repeated steps 1 and 2 two times.