Plating - Science topic

Rishibha Gupta Thank you so much for providing clear answer.

It is easy. You should watch the tutorial videos on YouTube. Use LOAD and BOundary condition in ABAQUS.

Hey Sofiyat, I would take a step wise approach: first isolation, second lipase activity confirmation. I’ll focus on isolation. If you have any evidence the fungi grow through the sample, then you could try break open the piece of butter or eventually cut a layer off with a sterile tool. Then you would take a small chunk of core butter and dilute it, probably with Tween80. Make serial dilutions (~3) and plate 100 uL of each in different media. Spread with a digralski loop or sterile beads. This should help isolating strains with different abundance in the original sample. I would expect these strains to grow very slow.

There will be some level of autofluorescence from blank cells without eGFP. This is to be deducted from your samples.

I have seen this article, where opaque zone around the colony is interpreted as weak proteolysis, and clear zone means strong proteolysis.

I would still wellcome some more information. It seems like I have opaque zone surrounded by a clear halo. Why would I have weak proteolysis near the colony and strong proteolysis further away ?

According to Pikovskaya R, Pikovskaya RI. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiol. 1948;17:362–70,

PKO liquid medium consist of NaCl 0.3 g, Glucose 10 g, KCl 0.3 g, Ca3(PO4)2 - 5 g, (NH4)2SO4 0.5 g, MgSO4· 7H2O 0.3 g, MnSO4 0.03 g, FeSO4· 7H2O 0.03 g, distilled water 1000 mL, pH 7.0–7.2, agar 15-20 g, autoclave (30 min).

How big is your HRM amplicon? And what are you targetting? A single SNP? Methylation?? From the melting curves, it looks like your amplicons might be quite large ??

I wouldn't use 2 month old plates unless there was no other option, personally.

Please read Busing-Levy articles; if you want, I can send them to you. They have developed an algorithm called, the Busing - Levy algorithm, which involves a lot of Matrix operations only. First, you should use a U Matrix called a Material Matrix. Then B Matrix. After finding these two matrices one can find out the angles for the h k l values and scan it for all the planes and finally collect the data and solve the single crystal structure.

You may look into the article here. Hope it helps

You may look into the article here. Hope it helps

10.1007/s13762-024-05635-3

We usually use the Bradford protein assays, both Bradford and BCA have advantages and disadvantages, but both are commonly used.

Hello Victoria Gillan . Have you got a solution for the matrigel dissolving issue? I am currently facing the same problem. I dissolved the cell pellet in a 1:1 Matrigel: Media ratio and allowed it to polymerize for 10 minutes in a 37C incubator by inverting the plate. When the media is added, the dome dissolved in some wells and becomes soft in others. When observed the next day, all the domes were dissolved. Kindly help me out.

If you're performing a self-ligation reaction and a control reaction (test) without any insert, and you're observing the same number of colonies in both, it suggests that the ligation reaction has occurred, but there hasn't been successful insertion of any additional DNA fragment (insert) into the vector during the ligation process.

Here are a few reasons why you might observe the same number of colonies in both reactions:

To troubleshoot and confirm whether the ligation reaction was successful but without insert, you can:

If you confirm that the self-ligation reaction occurred but no insert was successfully ligated, you may need to optimize your ligation conditions, ensure complete digestion of the vector and insert, and carefully monitor for contamination to improve the efficiency of your ligation reactions.

Hello Jacob Leighty,

Use the following protocol.

1. Determine the volume of collagen-I solution needed.

 2. Dilute collagen-I to 50ug/ml in 0.02M acetic acid to the needed volume.

 3. Add diluted collagen at 5ug/cm^2 surface area. The area of a well for a 96- well plate is 0.33 cm^2. To coat 5ug/cm^2 of collagen-I, you will need 1.65ug of collagen-I per well in a 96 well plate i.e.,(5 x 0.33/1=1.65ug). You will use 50ul volume to cover a well of a 96 well plate.Therefore, you will need 1.65ug/50ul for each well.

4. Incubate for 1 hour at room temperature.

5. Carefully aspirate the remaining solution.

6. Rinse well 3 times to remove acid, using PBS or serum-free medium.

 7. The plate may be used immediately or air dried and stored at 2-8° C for up to one week under sterile conditions.

Best.

Using topical cream as the source of antibiotic for the disk diffusion method may not be the most reliable or standard approach. The disk diffusion method typically requires the use of standardized antibiotic disks containing specific concentrations of antibiotics. These disks are manufactured under controlled conditions to ensure accuracy and reproducibility of results.

Topical creams may contain various other ingredients besides the active antibiotic compound, which could interfere with the diffusion of the antibiotic into the agar medium or affect the results of the test. Additionally, the concentration of the antibiotic in a topical cream may not be suitable for the disk diffusion method.

If you need to perform the disk diffusion method, it's best to use commercially available antibiotic disks that are specifically designed for this purpose and have been validated for accuracy and reliability. If you're looking to test the efficacy of a topical cream, it's more appropriate to use other methods such as minimum inhibitory concentration (MIC) testing.

Hello Rogerio,

In addition to staurosporine treated arms (25nM-100nM), do you also have a control arm that has not been treated with staurosporine ?

Is it possible that staurosporine is simply cytotoxic to your TC28a2 cell line and that’s why they are detaching?

staurosporine is commonly used to induce apoptosis in various cell lines. I’ve used staurosporine for that purpose in HCEC-1CT cell line( human colon epithelial cells) and some tumor cell lines( HUH-7 and NG108-15).

After treatment with staurosporine, cells become round and detach. Under microscope, you can tell that membrane integrity is compromised.

I understand that staurosporine is used as a differentiating agent in chondrocytes. However, there are some literature out there stating that staurosporine can also induced apoptosis in chondrocyte monolayer.

Hi Mohammad,

Thanks a lot for sharing your experience!

I switched to iPSCs after trying with MSCs for a long time, and keep failing. I usually did not have a problem with the iPSC-derived endothelial cells (iECs) on Transwells, but I was doing half-medium change each day. They were fine when I had to do whole-medium change for a permeability assay too, but I'm changing the media slowly, and never use vacuum suction on Transwells. Although this may explain why the MSCs were detaching right after the media change, I'm still clueless about why they would detach later despite looking fine immediately after a medium change.

We are culturing iECs as well as HUVECs on PDMS microfluidic chips, besides Transwells. For iECs, we coat the PDMS surfaces with 100 ug/mL Fibronectin and 50 ug/mL Collagen type I (this is the same recipe we use for Transwells), following a 1-hour surface activation with UV light (for HUVECs, the Collagen concentration is 100 ug/mL). I believe your Fibronectin concentration should suffice. The endothelial cells are indeed sensitive to medium change from what we have observed, so we do it as slow as possible. Despite this, for instance yesterday evening, most of my iECs had detached from the microfluidic chip that they were nicely attached yesterday morning (and they were under a constant flow of 4 uL/h, so I did not exert an extra force with micropipettes for medium change). There must be reasons, which we're not yet aware of, for the detachment problem, thus I'm looking forward to hearing from others too!

Best regards,

'Afternoon Vera,

To be fair, the typical 'nice' thermocouple or Pt100 thermometer has an intrinsic accuracy of +- 0.5 to 1.0 K.

<at least - can be higher!>

So if the display shows 99°C, there really isn't a physical difference if it were to show 100°C.

As to why there is such a large discrepancy (20°C!) between the target and actual temperatures, that is a puzzle.

Dr Suresh Kumar thank you for your contribution to the discussion

The movement of tectonic plates are primarly driven by the mantle convection, heat transfer and by the plume activity.

The convection is due to the radioactive elements decay releases the heat and residual heat left since the formation of the prototype planet which causes the matle material to become less dense, so that the less dense material rise and cold lithospherric slab sinks. This creates the continous cycle. The convection is the major contribution of heat trafnser whcich in terms creat the plate tectonics cycle.

The heat is also transfer by condction as well as radiation. But their contribution is minor. Rk Naresh

Are you vortexing your samples/dilutions right before plating? They will settle to the bottom of tubes over time, even when shaken.

But honestly, having less than 1 log of variability between replicates of the same condition when plating bacteria for CFUs isn't that uncommon or concerning. You'll almost never end up with 50, 53 and 49 colonies from three replicates.

If the QX200 Droplet Reader is not recognizing the plate during digital PCR (ddPCR), there could be several potential issues to consider:

Hi Subhrakanta Jena,

The pore size of the filter paper can be compared with the diameter of silica, and the one with a smaller pore size than the diameter of silica can be selected for filtration.

If you are using glass petri plates(autoclavable), make sure you choose a perfect pair otherwise if there is any space left in between then it could be a source of contamination.

What medium is in your plates? Are they LB or similar or a minimal medium?

Secondly, DH5a is a RecA- strain (as are most E. coli cloning hosts). These do not survive long on plates, maybe 2 weeks max. So I think everything is dead on your 6 week old plate.

However I don't understand why the frozen stock did not grow, which is why I asked about the medium you are using. You might grab a small chunk of the -80 stock and put in liquid broth to let it grow then streak out for single colonies. If you get it to grow then I would make a new -80 stock.

Your calculation of the combined electrode potential (E = E⁰_Zn -E⁰_Ag/AgCl) is correct

Thank you very much Hanh Hong Nguyen

Hello, remove kanamycin from nutrient media and see do you have a grow of culture or not, if you see grow it mens kanamycin resistance gene transfection or expression problem.

May consider using Permai fluorescence dye.

Malcolm Nobre thank you very much.

Hello Dr. @Gerhard Martens ,

you're welcome.

Thank you so much for your help, My question was solved by your answer.

Best Regards.

Nesa.

The magma becomes attached to crust as the magma cools. The attached crust moves with the magma as the magma is moves by the convection currents. The convection currents in the magma moves the attached crust of the tectonic plates. Convection currents drive the movement of Earth's rigid tectonic plates in the planet's fluid molten mantle. In places where convection currents rise up towards the crust's surface, tectonic plates move away from each other in a process as seafloor spreading. Heat from Earth's interior causes currents of hot rising magma and cooler sinking magma to flow, moving the crust along with them. Convection currents occur in the mantle due to the heating of the Earth's interior. The two statements that describe events that help drive tectonic plate movement are convection currents cause hotter magma to rise and cooler magma to sink and subduction plates cause cooling at convergent boundaries. Diverging boundaries are where the convection currents move upwards. Converging boundaries are where convention current move downward. When a ocean plate meets a continental plate in a convergent boundary the mantle current carrying the ocean plate is forced downward. The energy for all that movement comes from sunlight that is absorbed and re-radiated by the surface of the Earth and the rotation of the Earth. Atmospheric circulation, along with ocean circulation, distributes heat across the entire surface of the Earth, bringing us our daily weather and shaping regional climates. The reason we have different weather patterns, jet streams, deserts and prevailing winds is all because of the global atmospheric circulation caused by the rotation of the Earth and the amount of heat different parts of the globe receive. Heating and cooling of the fluid, changes in the fluid's density, and the force of gravity combine to set convection currents in motion. Heat from the core and the mantle causes convection currents in the mantle. This is how the heat is transferred, and how the earth's plates are able to move.Heating and cooling of the fluid, changes in the fluid's density, and the force of gravity combine to set convection currents in motion. Heat from the core and the mantle causes convection currents in the mantle. This is how the heat is transferred, and how the earth's plates are able to move.

The cell seeding density of 200-300 cells per 6 well is probably to low. iPSC like adjacent cells for better survival. You could try 48 or 24 well plates and/or more cells. The thawing AND freezing protocol should be optimized. iPSC should be frozen approximately 2-4 days after passaging. Freezing them significantly later after the last passaging, strongly decreases cell survival after thawing. Please find further information attached.

In a conductor, such as a metal, charges are free to move due to the presence of mobile electrons. When an external electric field is applied to a conductor, these free charges redistribute themselves within the material until they reach a stable configuration. This redistribution creates an electric field inside the conductor that exactly cancels out the external electric field.

However, it's important to note that this only happens in static conditions or when the conductor is in electrostatic equilibrium. In other words, the charges have stopped moving and there is no current flowing. If the electric field were not canceled out within the conductor, charges would continue to move until equilibrium is reached. This cancellation of the electric field inside the conductor is known as electrostatic shielding.

Phil Geis

Hi, I had not done this as this was a while back, but growth was seen originally on TSA which is seen In my graph, but upon re exposure of the same bacteria that had been re incubated in TSB back onto TSA didn’t promote growth.

Dear Tina Trinh ,

I would highly recommend to get in tough with the company asap, since they are stating this at the end of the link you have provided:

"Due to the sensitive nature of primary cells and cell lines, all quality related issues about the cells products must be reported back to us within ONE month period after receiving the products, no quality warranty (i.e. replacement of cells) will be provided after the ONE-Month period. Thank you for your understanding."

They should check their batch of the cell stock and give you maybe more clear thawing protocol.

We are usually thawing (different cells) like this:

Thaw the cells 30-60s with in a 37°C water bath (ice should/must still be visible which will keep the cells still cool). Than we are are transferring the cells into 10 ml pre-warmed medium I do use a pipet for that set to 800 µl. After 3-4 pipetting steps adding warm medium into the vial and removing than cells an the medium into the 10 ml reservoir, I spin the cells down (5 min 250 x g) aspirate the medium and plate the cells in new medium into the cell flask or dish. On the next day the medium is exchanged to get ridge of the rest of the DMSO.

Best wishes

Soenke

Hi,

Thanks for your responses.

Yes, these cells are grown in L15 media with 10% FBS, with P/S, in an incubator without CO2. I don't observe any changes in media colour/transparency, and the cells seem to grow normally, although a bit slowly (I think they are still adjusting to being thawed).

I will inoculate some culture on LB tonight to know for sure.

I believe you could use Poly D lysine

Hi. I've had them done when studying the protective effect of H. itama honey towards oxidatively stressed HEK293 cells.

The concentration of H2O2 I used to maintain HEK293 cell attachment to the flask is 100 μL of 1 mM. However, I recommend determining the IC50 of the H2O2 you plan to use in your lab, as it may differ from my conditions.

Typically, when inducing oxidative stress, cell death occurs, and as a normal observation, dead cells detach and clump together.

However, if you want to ensure cell attachment during stress, consider the following tips:

First, it's important to note that biofilm formation is a complex process that can be influenced by various factors such as bacterial strain, nutrient availability, temperature, and surface topography. Therefore, it's essential to optimize the incubation conditions for your specific experimental setup.

Here are some general tips for performing centrifugal filtration with Biofil 30kDa filters:

Prepare the filters: Before starting the filtration process, make sure the Biofil 30kDa filters are properly prepared. According to the manufacturer's instructions, the filters should be sterilized by exposing them to UV light for 30 minutes. Additionally, you can also perform a quick rinse with sterile water to remove any potential contaminants.

Choose the appropriate solvent: When it comes to dissolving the biofilm and transferring it to the filter, you have two options: NaCl or dH2O. Both solvents are suitable for this process, but they have different properties that may affect the outcome. NaCl (sodium chloride) is a common solvent used in many biofilm studies, as it has a lower critical concentration than dH2O, which means it can dissolve biofilms more effectively. However, keep in mind that NaCl may also affect the biofilm's structure and stability. dH2O (deionized water), on the other hand, is a more gentle solvent that may be better suited for sensitive biofilms. Ultimately, the choice between NaCl and dH2O will depend on your specific experimental requirements and the properties of the biofilm you're working with.

Dissolve the biofilm: Once you've selected the solvent, add it to the wells containing the biofilm and gently mix it with a sterile pipette. Avoid vigorous agitation or sonication, as this can damage the biofilm. Let the mixture sit for a few minutes to allow the solvent to dissolve the biofilm effectively.

Transfer the biofilm to the filter: Use a sterile pipette to transfer the biofilm-solvent mixture to the Biofil 30kDa filter. Make sure to handle the filter carefully to avoid any potential contamination.

Wash the filter: After transferring the biofilm to the filter, wash it with sterile water or dH2O to remove any residual solvent and debris.

Dry the filter: Once the washing step is complete, gently remove excess water from the filter using a sterile paper towel or a lint-free cloth. This will help prevent any bacterial growth or contamination.

Store the filter: Finally, store the filter in a sterile container or bag until further analysis. Make sure to label the filter appropriately to avoid any confusion.

If the blue and white colonies are similar size then it should be ok, a few extra hours in the incubator would let them get bigger.

How big is your insert? Was the plasmid mini prep good so that you say plenty of vector size DNA?

Remember that the dilution factor accounts for the dilution performed during the serial dilution process. For example, if you counted 150 colonies on a plate with a dilution factor of 1:100, you can calculate the CFU/mL for the original sample using the above steps1

Dear Malhar Desai,

Your using protocol is correct. But you do must without PBS washing. Both set up give best results.

Thank you

Based on my experience, doing this through Mathematica is pretty straightforward. You need to use the "VariationalMethods`" package. Check the following link for details.

Thanks for the information. Sounds like you have a problem with the ligation reaction itself.

Here are a few things you might want to try different (and what I have always done with success):

1. Perform the ligation at RT O/N.

2. Cut the amounts of vector/insert way back - use 1ng of vector and then adjust your vector ratios based on that.

3. Increase you ratios to 1:40, 1:50, 1:60 - never found the 1:1, 1:3, 1:5 ratios to work consistently.

4. Use 1ul for transformation.

5. Resuspend transformed cells in 250ul SOC and after incubation plate the entire amount.

To seed 0.5x10^6 cells per well in a 96-well plate (but only 5 wells) and considering you have a T25 flask with a cell density of 0.6x10^6 cells/mL, you can follow these steps:

1. Calculate Total Cells Needed:

0.5×10^6 cells per well x 5 wells = 2.5×10^6 total cells needed.

(2.5×10^6 cells): (0.6 ×10^6 cells/mL)=4.17mL

This is the total volume of the cell suspension needed to get the required number of cells.

Take the cell suspension, centrifugate at 2000 rpm for 5 minutes and remove the supernatant of the existing media (to make space for the cell suspension).

Add 4,17 mL of fresh media to the cells pellet and gently mix the cells to ensure a homogeneous cell suspension.

Dispense 0.83 mL (830 μL) of the cell suspension into each of the 5 wells in the 96-well plate.

Place the 96-well plate in the incubator for cell attachment and growth.

Ensure that you maintain proper sterile techniques throughout the process to avoid contamination. Additionally, be mindful of the timing and conditions required for the specific cell type you are working with.

Dear Corinne,

I am having a similar issue. I wonder, what was your conclusion for this situation?

Hello Gage,

For your application, you will need to consider the growth support topography and growth culture conditions in terms of surface area and volume of culture medium available to the cultured cells.

The V-bottom wells are not ideal for your application because as you mentioned, it will not provide enough surface area for the protein to interact with the spleen cells, since the cells will settle at the bottom after some time and cell activity will be significantly lowered. Moreover, in V-bottom wells, the cell morphology will also be affected as cells will appear to aggregate in the centre of the well. This is more relevant if cells are adherent in nature.

I agree to what you mentioned regarding

incubating the cells and protein in flat bottom wells and then transferring them to V-bottom wells right before centrifugation.

During the 72h incubation period, do not mix the cells and proteins with a multichannel one or more times because this action of yours will disturb the interaction between the cells and the environment, and you will be doing more harm than good. Cell and protein interaction will occur within the wells due to the Brownian motion of protein molecules. You need not worry about that.

The article attached below will be helpful!

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662085/ 

Best.

amino acids are polar molecules i think you need to change stationary phase to C8 or C18 of your plates

@Michael J. Benedik

Thank you so much for answering my question! The recipe for TSB media is 15 g/ 500 ml of water (suggested by sigma), and that for TSB plates is 6 g of TSB + 3 g of agarose in 200 ml of water. The agar concentration is 1.5%, and the TSB concentration is the same for TSB broth and plates. The only thing I am worried about when making the plates is that I didn't put the agar flask into a 56 oC water bath after autoclaving to lower the temperature. But I will definitely make a new batch of TSB plates and try again!

Zuffain Hussan Can you share your Abaqus model (.inp)?

Malcolm Nobre

Thank you very much

I learned a lot

In plate heat exchangers, the heat is transferred between two fluids though a conductive plate. There, convection and conduction are the heat transfer processes between the fluid and the plate, and conduction is the process through the plate itself. Since convection is much more efficient when the flow is turbulent, high Reynolds numbers (>4000) are looked for.

The same is needed in heat pump heat exchangers, since the heat is transferred from the hot fluid to the heat pump gas through the walls of tubes.

What do you intend to stain: cell surface markers, an intercellular protein, organelles, nuclei, nucleoli, ... ? Without knowing any details of your experimental set-up it is impossible to give you any advice; do some research on your own and then ask such more specific questions.

Denis Benasciutti That only applies to steel bars in reinforced concrete; in this type of structure, the shear load at the interface is transmitted through friction, which is somewhat lesser, and through shear studs. Additionally, I need the plates to detach from the concrete when they buckle, so I cannot perform an analysis with rigid node-to-node contact, which forces me to use surface-to-surface or general contact.

Dear Alaa Kaoud

The detachment of cells after replating into a monolayer can occur due to various reasons, including:

To avoid cell detachment in the future, consider the following strategies:

By implementing these strategies and optimizing experimental conditions, you can minimize the risk of cell detachment and improve the reproducibility of your experiments. Additionally, troubleshooting specific issues encountered during cell culture can help identify and address underlying factors contributing to cell detachment.

Hi Vinay,

You can measure the melt Temperature (Tm) together with the glass Temperature (Tg) as well as the G'/E' G"/E" and Tang (delta) of polymers with a Dynamic Mechanical Analysis (DMA) and Differential Scanning Calorimeter (DSC). The measurement of these parameters should tell you pretty much everything about the rheological properties. DMA more accurate measurements but is more expensive.

I have done tests longtime ago as a part of my Masters thesis but the principal is the same.

Below is an article I have found that described how these measurements are done as well as the explanations of the results. You may also take a look at the references the publishers have cited.

Ignace

Pharmaceutics. 2010 Jun; 2(2): 78–90.

Published online 2010 Mar 24. doi: 10.3390/pharmaceutics2020078

PMCID: PMC3986708

PMID: 27721344

Assessment of Thermal Transitions by Dynamic Mechanical Analysis (DMA) Using a Novel Disposable Powder Holder

Mohamad G. Abiad,1,† Osvaldo H. Campanella,1 and M. Teresa Carvajal2,*

Author information Article notes Copyright and License information PMC Disclaimer

Go to:

Abstract

Foods and pharmaceuticals materials are exposed to various environmental conditions during processing and while in storage; therefore, stability and quality are key attributes of concern. The properties of foods and pharmaceutical materials that define their quality are affected by conditions such as temperature, humidity and time. Glass transition is considered a key material property to understand how these external conditions affect the stability and quality of foods and pharmaceuticals. Thus, investigating the thermo-mechanical properties of these materials as well as characterizing the glass transition temperature have a great interest not only in the food industry, but also extend to the pharmaceutical and polymer industries. The aim of this study was to design and test a new disposable powder holder that allows the use of a dynamic mechanical analysis (DMA) instrument to test and characterize loose powder samples. The disposable aluminum powder holder was designed and constructed to be used in the single cantilever configuration on a TA Instruments RSA III DMA. Three different powder samples – Felodipine, polyethylene-oxide (MW 900 kDa) and HPMC (E4M) – were used for validation. The use of this powder holder allows the detection of different thermal changes of powder samples without compacting and when large sample size is necessary for detection and/or interpretations

The fact you have 2 sizes of colonies is not good, I would use 100ug/mL Amp on your plates to increase selectivity for your key plasmid, this should eliminate the differing sizes.

Have you considered using autoinduction method with Terrific Broth, Studier F.W. et.al.

This is a super rich media that can get 14 OD600 cultures, when used in baffled flasks, when the glucose runs out as a food source then lactose becomes available once the cells are at a good OD and induction kicks in.

Hello Kelly Smith,

For differentiation of SH-SY5Y cells, usually 10μM all-trans retinoic acid (RA) is used namely, DMEM containing 1% FBS + 10μM (RA) for 72 hours to induce differentiation.

You have 10μM RA which upon addition to each well will get further diluted in DMEM + 1% FBS. You need to have a stock solution of a higher concentration. So you prepare a fresh stock solution of RA (say 5mM) and use the below formula

C1V1= C2V2

Where

C1 = concentration of stock solution 5mM

V1= volume of stock solution required (say X)

C2= working concentration (10μM)

V2= volume needed for the experiment (50 wells x 100ul) i.e.,5000ul if you use 100ul per well.

5000μM x X =10μM x 5000ul

X = 10ul

You may add 10ul of 5mM stock RA to 4.990ml of media (DMEM + 1% FBS) to give 5ml of media which you could use for differentiation by adding 100ul per well in 48 wells.

I have attached an article below which may be helpful.

Best.

Hello Ethan,

As per the information provided by you, there could be two possiblities.

1. You may have wrongly counted the cells while plating, or

2. You may have wrongly concluded the confluency state of cells.

I would infer that 15cm dish with 5e6 seeding density may have become overconfluent on Monday because of which there is lack of space for cells to grow further as well as the media may have been depleted of nutrients.

On the other hand, the 15cm dish with 3e6 seeding density may have almost reached confluency with some space left for cells to grow besides the media still being fresh and nutrient- rich.

I would suggest you plate less cells this thursday (say 3e6 so that it reaches 80-90% confluency after 4 days i.e., Monday/Tuesday and 1.5e6 cells so that it reaches near confluency at one/two days later i.e., Wednesday/Thursday) with the SAME amount of media in both the 15cm dishes.

Best.

If you are trying to isolate colonies on the plate it appears that your original inoculum has too high of a density. Dilute it in half, and then half again, and maybe again and then do your pour plate or streak for isolation so that you can get isolated colonies for your pure cultures.

If you still get the right size bend (showing that your plasmid is in) I would not worry about primer dimers

Your enumeration of microbes as Colony Forming Unit ( CFU), you should be very perfect what type of microbes you are dealing with. Accordingly you should proceed serial dilution pour plate or spread plate technique. For instance, if you are dealing with bacteria, your accurate dilution will be 10‐⁴, 10-⁵ and 10-⁶ . Suppose you get 30 colonies in 10-⁵ by inoculating 0.1 ml, Then your CFU will be average count x dilution factor and find out as CFU per ml or per gram as follows: 30 x 10⁵= 3x 10⁶ for 0.1 ml So CFU per ml will be 3 x 10⁷. It is better to express as log value. Your answer is 7. 477. Why log value? Because exponential growth of bacteria is always in log phase. If you deal with Actinobacteria , go one step lower dilution I.e., your sampling will be 19³, 19⁴ and 10⁵ ( because Actinobacterial population is less than bacteria in normal soil). Similarly, when you deal with fungi, choose the dilution 19², 19³ and 10⁴. Hope, I could clear your doubt.

One easy attempt would be to reattempt just as you described but make sure your antibiotic selection reagents are new. Kan and G418 can break down over time. If you still have your transformed bacteria vial, just spread that on the new plates to not waste anything.

Hey Alaa A. Rashwan , I think u should think about the following:

1. Cell singularity: make sure when u trypsinize the cells, they become single. and if u centrifuge afterwards, make sure when u reconstitute in media, u pipette properly to ensure the cells are single again. Having cell clumps (>3 cells) might cause this difference

- P.S. avoid aggressive pipetting as it might shear the cell membrane and decrease cell viability

2. Cell Seeding: make sure u seed the same number of cells in each well. i.e. u have proper pipetting techniques. aspirating extra or less volume might contribute to such differences.

3. Cell Detachment: when u add drugs/PBS/MTT to the wells, make sure the pipette touches the wall and don't release the liquid in the center of the well as this might was away some cells.

4. Media aspiration: if u r using an aspirator to remove the media, make sure u don't aspirate the cells as well by doing the following:

a. decrease the pump pressure to be the least.

b. don't touch the bottom of the well. this would increase the chance of cell aspiration

c. monitor ur aspiration technique by checking the wells before and after the aspiration

Hope this helps.

Erika Avila

I met the same problem! Have you figured this out?

Hello.

Your cell culture probably infected by mycoplasm.

Read about cell line contamination by mycoplasm

Yes it is necessary to include a housekeeping gene for each plate, and calculate expression values based on that. Cannot extrapolate values for housekeeping genes as each PCR run is different due to small pipetting errors, machine readings, reagent thaw/freeze cycles etc

To quickly distinguish between live and dead C. elegans on a mixed NGM plate, you can use a technique called "gentle touch." Live worms will respond to touch by moving, while dead ones will remain motionless. Gently touch the worms with a platinum wire or an eyelash pick, and observe their response to identify the live individuals.

Are you trying to express your protein of interest? In that case, you might need to use BL21 or some other expression vectors. DH5a in not a protein expression vector, so your pET28a containing the gene of interest might not get expressed, thus everything is growing on the plates!

Hi, I found out, that they offer qBase+ for free https://cellcarta.com/genomic-data-analysis/, how did you solve your problem?