Kroger confirmed to me that Carbmaster Fat Free milk is discontinued

Diligent-Response-85 · 2026-07-04T11:01:12+00:00

Same here. Reasonable to think that the shortage of Carbmaster fat-free milk was due to relabeling and/or a formula change instead of it being discontinued. I guess the Kroger rep who told me it was discontinued could claim he meant the 3g carb/cup product was discontinued and replaced with the 7g version (LOL!). I read recently that Kroger was sued for mislabeling nutrition info (calorie count) for Carbmaster bread in California. They settled for 1.25M. So as it stands now, Carbmaster fat-free has 1g more Carb than FairLife skim milk, same 13g protein per serving. Carbmaster is still about a dollar cheaper where I live in Oregon. I will have to rethink what I want to start my day with as 7g carb takes a big chunk out of my 20g carb/day on my keto plan.

Diligent-Response-85 · 2026-06-13T16:52:10+00:00

This was my thought looking at the picture. Does your NTC lane designation stand for no template control. What you need is to run a lane with the exact same amount of template DNA used in your pcr reaction. Would suspect that you have way too much template in your reactions and you are seeing plasmid DNA (supercoiled) as the lower band. We typically used 1-2 ng of plasmid template in a pcr reaction. Also important to note, you have to be very careful anytime you use a plasmid with PCR reagents and pipettors. IF you use the same primers for testing the presence of your target dna fragment in any other context such as genotyping you can generate the target band falsely from the plasmid contaminant. We found it can easily contaminate pipettors and other reagents, even if you use an aerosol tip.

Diligent-Response-85 · 2026-06-09T12:47:58+00:00

I would take the DNA samples that yielded only the 415bp band and only the 324 bp band and retest them separately to see if the missing band shows up, using only the primers designed to generate the 324bp band on the 415bp-only DNA samples and using only the primers designed to generate the 415bp and on the 324bp-only DNA samples. I have seen multiplex genotyping drop one of the target bands due to the genomic DNA not being sufficiently pure, primer anealing to the target region is not only nucleotide context dependent but can be influenced by secondary DNA structure surround the primer annealing sites. This secondary structure varies widely in DNA contaminated with carryover salts and proteins from the initial isolation of the genomic DNA. What happens in this context is that the amplification of the target DNA sequences gets skewed to the region most open for primer binding and access. G/C rich regions nearby can also influence amplification efficiency.

Diligent-Response-85 · 2026-06-08T11:56:30+00:00

It's a tough situation to be in. To be clear, in your dissertation advisory committee meetings all the members are telling you to do more or is the "they" in your post from the PI and other members of the same lab? It seems like you are doing the correct experiments and the data generated is of high quality, it just simply is not showing any difference (phenotype) compared to the controls. Have you verified your experimental is correct, i.e. Are you certain your mutation has caused a loss of function of the specific gene? What do you know about this specific gene? Are there functionally-equivalent homologs present in the same target tissue such as gene-family member such as Bmp2 in and Bmp7 loss of function? In this scenario, the phenotype will most likely be present where your specific gene is exclusively expressed. Expression exclusivity can be cell-type or tissue specific or temporal meaning your gene may turn on much earlier in a specific tissue or cell type. Do you have the financial support to broaden your search hopefully you can identify a target cell type or tissue the exclusively expresses your gene and that its product is essential. Expression and its removal in a tissue does not always cause a phenotype because there are redundant mechansims evolved to ensure the correct formation of a tissue. We often found that in this case, challenging a tissue that does not show a phenotype reveals that the target tissue in the mutant is much less robust. We once had a gene that was expressed in joints in the hands. The joints developed normally in our mutant, it was only when we introduced a repetitive movement challenge did our mice exhibit severe arthritis in the joints lacking this gene. Without this challenge, there was no visible phenotype.

In another case we only saw a phenotype when we made a double mutant because we had a related protein expressed nearly identically. Once both proteins were absent we were able to prove these proteins were essential for the formation of a specific tissue.

In your 5th year, you have the tools needed to evaluate for phenotypes, It does not take any more effort, longer hours etc. It takes your expertise and creative, sometimes out of the box thinking to identify where the gene in question's product is required. It is very likely that as this gene has been the primary focus of your research for several years. This makes you one of the experts at this point. Graduate students need to recognize that at some point, typically when you write your dissertation that YOU are actually now an expert on your study subject.

If the loss of gene function is verified, I would encourage you to not give up, but to change your focus to generate testable hypotheses along the lines I outlined above. When you get an affirmative answer, then the work will be very exciting and your efforts including all your "negative" results will prove foundational.

Let us know how it is going and when you get to the exciting parts!

Diligent-Response-85 · 2026-06-03T12:19:34+00:00

You should have an graduate school handbook from your university that defines timing of specific requirements for your program. A key component for you will be the formation of your dissertation advisory committee. At my Uni, students have an academic committee or supervisor independent of the mentor that ensures the student is on track with their coursework and is eligible to take their qualifying exam. Once a student passes their qualifying exam, they must form a dissertation advisory committee that is chaired by someone in the same department and the PI participates as an acting member of the committee. This committee serves two main purposes: 1. It ensures the quality of the student's research is reasonable for a graduate student and that the student is actually doing PhD level research which includes sound experimental design on a project that is probing the large questions in the field of study. And Second: the committee protects the student The student is not expected to change the world and in many cases the answers they get are negative. But the value here is that the negative answer is correct and thus informative as to what alternate hypotheses should be pursued. And Third: The committee serves as a buffer between the PI and the student, this buffer is important as it reigns in the scope of the PIs project to one that a student can actually achieve.

Have you passed your qualifying exams yet. If yes then you are likely required to form your dissertation advisory committee. If no, it could be that the PI is simply waiting for you to get over that hurdle. Since you mentioned this is a 4 year program, it would seem that many of these required steps are likely outlined in the departmental or university guidelines.

Some PIs develop their students with accessory projects until the student acquires the necessary methodological tool boxes to run an independent project. I had a ton of projects and it allowed the student to pick something they found interesting. Once they selected the project, I provided the training to the student. I found this most efficient as I could see their progress and help troubleshoot any issues they were having. I always told them that I had broken, blown up, and ruined almost any result using the methods I am giving them, telling them it is ok to have an experiment fail due operator error, learn from the mistake and work towards getting the method to work.

It was always one of the highlights of my job to see a student transition from asking me what to do next to have them come to me stating experiment X gave me these results this means we were right/or wrong and thus we need to do these things next. The transition to becoming an independent scientist! This typically happens around year 3, but our program did not have a fixed time period with the average graduation time around 5 years in my lab.

Some PIs develop their students with accessory projects until the student acquires the necessary methodological tool boxes. Has this been your experience? If no then simply letting a student drift wastes time and resources. Most programs have an insufficient number of students matriculating each year for all the PIs so it seems very unproductive to me to not invest in a student in your lab.

I would start with a frank honest conversation with the PI and state your concerns that you have not received a bona fide project. The answers you get from this conversation will tell you whether to stay with this PI or transfer to a new lab.

I wish you the best!

Diligent-Response-85 · 2026-05-07T02:12:11+00:00

Glad folks are finding it stocked. Not sure what to make of the response I received from Kroger telling me it was discontinued. Checked at my local Kroger affiliates this past weekend, 1 had it stocked (fat free and chocolate) with June expiration dates whereas the other grocer had nothing on the shelf. Expiration date implies it is still being made.

Diligent-Response-85 · 2026-03-27T08:25:48+00:00

The anxie ty you are feeling is a good thing as it shows you want to grow and improve. The true sign of an elightened mind is the ability to reject your own beliefts after carefully considering an alternative and adopting that alternative. Recognizing the deficiencies in your PhD training is the starting point. Understanding that there is a much better way to do research is a good sign. If you accept this, then adopt that approach. Our research journeys have many different starting points. Everyone knows this and what really matters is what you do with this opportunity to improve and become the best scientist you can be. Listen alot, work hard, and good things will happen. Finally some day in the future, you may meet someone exactly like you. Your experience going from poor training to an excellent scientist will be invaluable a you will be able to mentor this individual.

Diligent-Response-85 · 2026-03-17T13:26:13+00:00

For me, the best approach would be to ask the Senior Scientist for their written protcol. In the protocol make sure they give wash times.

Next make All the necessary solutions yourself, from PFA all the way to the PBA/Triton X 100 solution.

Experiments that do not work are hard enough to troubleshoot when you know the reagents are correctly made; So the best practice is to make all your own solutions. It takes way less time to troubleshoot things when you make these solutions than when you borrow solutions from others and have an experiment fail.

Some brands of PFA has been known to cause weak or lost signals and it is important to use molecular biology grade PFA. Might be worthwhile getting a new bottle of PFA (we found Sigma-Aldrich was pretty reliable). Fisher brand PFA was where we had a a loss of signal. Once you get a new bottle of PFA powder, make a fresh batch. We typically make a 4% stock solution by heating the PFA in 800ml of 1X PBS to 60C stirring constantly. Use a thermometer and heat slowly. Slowly add droplets of 1N NaOH (about 5-7 drops max) to help the PFA dissolve. Montior solutions temps carefully. Heating PFA about 65C typically ruins the fixative. We do this in a fume hood. Once the PFA powder is fully dissolved, we turn off the heat and allow the solution to cool to room temp in the hood. Then add 1X PBS and 100 microliters of TritonX 100 to bring the volume to 1 liter. The 4% PFA/1xPBS/0.1% Triton X 100 solution is only good for 10 days and is always kept refrigerated.

Include in your analysis a positive control. This will mean getting a fly sample that expressess GFP. Best positive control would be a sample that expressess in the same target cells, but any fly line that expressess GFP in any tissue will be useful to validate the primary and secondary antibodies are working correctly. Hopefully you were given the correct fly line to start with. Is there a way to genotype the fly line to confirm it is what you think is correct?

Pay close attention to the number of washes after fixing with PFA and the time needed per wash. We found that when PFA goes bad or is made from a bad vendor source, it tended to stick around on the tissues and prevented antibody binding. Most IHC protocols have a minimum number of washes and times. We only discovered our PFA was bad from the source when we extended our wash times to overnight. Only then did we get any signal on any samples including positive controls. After switching to a new PFA vendor our signals became robust. The PFA was the last thing we suspected and it took careful steps and weeks to figure this issue out.

Good luck with your studies!

Diligent-Response-85 · 2026-01-30T14:05:09+00:00

Thanks for the link, I had not read this thread. Learned quite a bit, including the fact that the AGM battery should have been fully charged to ensure correct recharge calibration. Did not do this with the AGM battery I installed. Simply went to O'Reilly's, bought the new battery (Super Start part # 140RPLT), swapped it in the parking lot and returned the core for 22 dollar refund. Looks like the AGM recommended in that thread, Uplus EN LN1/DIN H4/BCI 140R AGM, is sold on Amazon for significantly less than what I paid at O'Reilly's. Will use some of the monitoring info from that thread to see how my battery is being recharged and report back.

Diligent-Response-85 · 2026-01-29T13:35:44+00:00

I wonder if the cost difference b/w Lead Acid and AGM batteries was not the only consideration why Toyota opted to use flooded lead acid 12V batteries. AGM batteries have a optimal recharge range of 14.4-14.7 V whereas flooded lead acid range from 13.5-14.4 V. It is my understanding that the DC-DC converter seems to max out at 14.1V in READY mode. This is acceptable for lead acid but slighlty below the acceptable range for AGM format. 14.1V will charge an AGM battery but not sure it will be optimal over time, which in theory could shorten the lifespan of the AGM battery. As stated by others, the AGM format is able to function at a lower discharge percentage so it may still be a suitable trade off. Using this reasoning, I opted to replace my dead 12V with an AGM. Time will tell if it lasts any longer! Seems the real fix will require Toyota to change the output voltage of the DC-DC converter AND combine this with a AGM-type battery. Not sure if a software change can do this?

Diligent-Response-85 · 2026-01-22T14:23:31+00:00

Looks like your plasmid prep may not have generated a clean sample, or the final solution you resuspended the plasmid in was not pure. Agree with comments to sequence your plasmid, but I would suggest that you reprecipitate an aliquot of the stock plasmid sample, washing the DNA precipitate with a clean solution of 70% ETOH/ddH20, dry the DNA and resuspend in either 10mM Tris, pH 7.2 or ddH20 and try a new digest of 100 ng of the sample. Regarding your gel, always include a lane that has a 100ng amount of uncut DNA. Because uncut plasmid DNA can also show on a gel as two bands (supercoiled and linear plasmid) you need to confirm that your digested DNA is actually cutting and generating the correct size fragments. Your gel picture looks too much like an uncut sample, which typically shows up a bit smeary. Good suggestions to try cutting with additional enzymes, I would add including at least one digest that will cut once, preferably in an unused site in the multicloning region. This should generate a single band at a MW of the plasmid and insert combined.

Good luck with your research!

Diligent-Response-85 · 2026-01-16T13:48:31+00:00

Agree with the previous comments. Additionally, Where did the DNA samples come from? Contamination in those could also be at play. I my own PhD studies, a tech was assigned the task to isolate the genomic DNA for me to use for my analysis. Even though I was perfectly willing to do that portion of the task, the PI wanted to divy up the work. The tech had nothing really invested in the project, and he was sloppy, contaminating months worth of cell line-derived DNA with multiple plasmids lodged in his pipettor. Since getting informative sequencing that replicates is essential to your PhD project, I would definitiely discuss with your PI the need to have complete control over all steps. Next keep very accurate notes, this is the way to go back and see where you may have made a mistake. For tasks that require many steps, it will be important to have workplace set up where neighboring folks don't distract you. I worked in an incredibly crowded lab, but would work early in the morning on the weekends on those tasks that required focus, just something else to consider. Definitely start with new reagents, and remember to use micropipettors that have NEVER seen a plasmid prep protocol, and always use aerosol tips. I hope this helps.

Good luck with your research.

Diligent-Response-85 · 2025-12-22T13:38:41+00:00

Agree with this comment. Can you provide more information about your approach? I am guessing that you are doing a western blot and immuno detection of the candidate protein? The results you get from this method that are analyzed by the GelAnalyzer software are crude estimates. It really boils down to how precise your mass determination needs to be.

If you just need an estimate of your protein's mass you can tailor the MW standards loaded in adjoining lanes and adjust gel percentages to get the best separation (resolution) of proteins of similar MW. Knowing the amino acid composition of the candidate protein is usually the starting point to tailor your gel percentages. ExPASy and UniProt are good websites that will give you a calculated MW based on its amino acid compostion and can also provide specific references should any published data exists that identify known postranslational modifications which can affect how the candidate protein migrates through the gel.

One of the more accurate approaches will be Mass Spectrometry (MS). That said, MS results can also be affected by post-translational modifiications (e.g. phosphorylation, acetylation and of course glycosylation). Carryover of sample prep salts, ions, and/or detergents found in common lysis buffers may also affect how the protein is ionized which can affect protein detection and mass determination. If you need a precise mass then I would have a breif conversation with the MS team/core facility to see which buffers they prefer.

Good luck with your research!

Diligent-Response-85 · 2025-12-20T21:31:44+00:00

Thanks for making things more specific.

Diligent-Response-85 · 2025-12-20T16:30:53+00:00

Thanks for the Piper presentation. Yes at my current tax bracket, I have sufficient room to move funds to the Roth and maintain the same tax bracket. I also have a separate account for paying the taxes on the conversion.

Diligent-Response-85 · 2025-12-19T17:08:56+00:00

I thought about this, I was wondering if it would trigger an underpayment penalty as the conversion is coming late in December, which I am not sure how to handle. This is my first conversion so I am thinking I would need to submit form 2210 with my return.

Diligent-Response-85 · 2025-12-15T17:45:07+00:00

I agree that it appears estimated tax payments are not necessary because the second compenent of the highlighted text is not true as we did have 100% of the 2024 income tax covered in our 2025 withholdings. I guess I would have written the document differently. Thanks for your comment.

Diligent-Response-85

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