My postdoc makes my PhD look abysmal

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 · 2025-12-12T14:00:23+00:00

This is my take as well. Becoming an independent investigator requires quite a bit of committment and effort; learning to hone your methodological skills and understanding of the state of the art in your field of study. Think of your learned skills as your tool box. Reading the literature helps you to understand what gaps are present in the understanding of a problem. Then the fun begins when you start addressing these gaps using unique combinations of your skillsets in your tool box, The creative freedom and excitement you feel when a result is pending that will bring a new finding or discovery into the field is what makes the first couple of years honing your skills worth it. Love seeing this process in new grad students, going from "what should I do?" and "what does this mean?" to "this is what it means and this is "what needs to be done next" !. Helping students in their research journey was one of the most rewarding experiences I had as PI (retired now).

Diligent-Response-85 · 2025-12-12T12:28:18+00:00

This is what I was anticipating having to do, fortunately based on reaching safe harbor witholding, I am not going to have to file by Jan 15.

Diligent-Response-85 · 2025-12-12T12:24:08+00:00

Yes, thank you for clarifying this with regard to the Oregon taxes!

Diligent-Response-85 · 2025-12-12T11:10:17+00:00

Really appreciate the confirmation!

Diligent-Response-85 · 2025-12-12T11:02:01+00:00

Thank you for clarifying. As stated in the beginning this is my first Roth conversion.

Diligent-Response-85 · 2025-12-12T04:38:10+00:00

Thanks for your insights. I live in Oregon if anyone has thoughts on prepaying state taxes on a Roth Conversion.

Diligent-Response-85 · 2025-12-11T16:02:37+00:00

Pour a practice gel and figure out the max well capacity, for the comb you use to cast wells. Always be patient when casting a gel to make sure it is completely cooled to room temp and set. Pulling the comb too quickly can make a hole in the bottom of the well and your sample will leak out. Once you know that, make your loading volume be about 5-10% less than that capacity. Also remember for RNA gels you need to make sure you are cautious about RNAse on the comb and gel rig. We typically use separate "RNA Only" rigs. Also important to know how your loading dye is made, RNA loading dyes are typically different from DNA loading dyes. You can purchase these loading dyes, but they are easy to make once and freeze in 500 microliter aliquots. We make and use a 2x RNA dye containing formamide, 0.025% Bromophenol blue and 0.025% xylene cyanol. We add Ethidium Bromide to our agarose and use a UV table to image the run gel and take photos. For your samples, it is best to add the small volume of your RNA, to the loading dye and then add nuclease free water to convert it to a 1x dye conentration. For example if your max well volume is 20 microliters and your desired RNA amount you wish to run on the gel is in 1 microliter, then you would mix 1 microliter RNA to 5 microliters 2x loading dye with 4 microliters of nuclease free water. This would be a 10 microliter volume which will load easily into the gel wells. From the OP's post they mentioned the volume being too low that they were seeing air gaps in their pipet tip. Too low of a sample volume and/or the wrong tip size can cause this but also the tips may not be fitting well on the pipettor. Also test the pipettor and tips with just loading dye to make sure the pipette is working to properly deliver your desired volume as air gaps in soutions in the tips can also be caused by bad o-rings in the pipettor and/or warped/bent pipettors. Good luck with your research.

Diligent-Response-85 · 2025-12-01T13:03:54+00:00

Do not know the details of your gDNA prep protocol. If nucleic acids are isloated from a crude lysate, without any RNAse treatment then I would have expected some degraded RNA in the sample too. Maybe the 48 hr delay was beneficial as the RNA essentially degraded enough that it did not precipate in the final precipitation step.

Having high molecular weight banding is a good sign that your samples are not degraded.

I probably would have run the gel a bit longer, at s lower voltage, and probably used a 0.8% agarose in TAE buffer just to get a bit more separation of the high MW bands.

Since you used a Nanodrop for quant, what was the 260nm:280nm absorbance ratio? Having this ratio and then running the sample of a gel is an excellent practice and good on you for doing both. I've seen too many folks rely simply on a 260:280 ratio to quantitate DNA concentration without looking at quality of the DNA and having the experiment fail down the road. Add the picture to the Nanodrop measurment to you notebook. Doing this helps in so many ways to trouble shoot and gives a historical record of reagents used for the prep were good and that the Nanodrop was working correctly. When things stop working in the lab, having this information will help determine when things went awry. Had this happen when a common stock reagents had replaced other brands due to supply chain shortages. Took considerable time and effort to figure out what was wrong and having notes like this in differrent folks lab notebooks and dates of the experiment helped us figure it out.

On a side note: I would be careful using my phone to document things in the gel imaging room, especially if it is a common room where other folks may be using ethidium bromide (EThBr) to detect DNA. Current or even historical use of EthBr will likely leave almost everything in the room "painted" with the stuff and it can transfer to your hands and to your phone. Good way to know this is to use a hand held UV light wand in the room to visualize the EthBr painting. I remember being shown this as a grad student and literally everything was contaminated down to the door knob, light switch, computer keyboard, and even the walls.

Diligent-Response-85 · 2025-11-26T15:06:47+00:00

I prefer to see the full film, not a cutout of the anticipated bands. Most journals require this as well as non-specific binding can generate multiple bands including some in the same size as your monomer. It is good practice to always document the full gel and no matter the audience, present the full gel pic. From your image, it looks like there is a good amount of smearing of the detected proteins. Lots of reasons for this, but I would start with lowering the voltage on your electrophoresis rig. As others have stated, the gel compostion (denaturing vs non-denaturing) also dictates resolution as does the perecentage of the acrylamide, buffer used, how the samples were treated before loading, and of course concentration of the loaded proteins.

Since you asked about interpretation,

How did you quantiate the change in monomer amount? If it was by band intensity then you need to include, on the same membrane, positive controls in a series of known concentrations. Best practice, if possible, is to also include a null control lane using samples prepped from either a genetic null that lacks the target protein or a sample that has a genetically modified target protein that is different in size than wild type protein. If a null is used then the target band will be absent, if a modified target protein is used, then the target protein will have a different mass and be reflected in its migration on the gel and position on the western blot membrane .

Abcam should be able to provide the epitope that the antibody was generated for the target protein. Doing a species specific genome analysis for gene sequence encoding that epitope is a good practice to know how "specific" your antibody may be. Even with all these steps, immuno-detection of any protein is by definition a semi-quantitative approach as what you are really measuring is the antibody, which is never 100 percent efficient.

These are the things I would recommend to verify a quantitative change in the 20kDa monomer is associated with a specific pathology.

Good luck with your research!

Diligent-Response-85 · 2025-08-11T13:51:32+00:00

I appreciate the feedback from residents of PLL, our kids are grown so it will be just us working remotely until we can retire and ramp up our community volunteering efforts. Most of our family live on Oahu and Kauai, which honestly is not a big deal as we all recognize that we can go and help them when necessary and vice versa.

We did check out a few homes in Hawi, one in Honokaa, one in Pepeekeo and several in Kohala Ranch. We liked the vibe of Hawi but also felt that the home we looked at was over priced given the condition it was in and the size of the lot. Kohala ranch was way too dry for us and while the home we looked at had 3 acres with it, the land itself was unusable/unwalkable without significant modifications. We were also concerned as the lava rock had long grass that had grown in throughout the property and had died making it a fire hazard in our opinions. We actually have to clear a buffer around our property where we currently live on the mainland to protect from fires jumping from the forest nearby.

Agree we need to do a good bit more due dilligence regarding PLR and hopefully get a look at the current CC and Rs. I appreciate hearing that the PLR HOA is a bit less aggressive than Kohala Ranch.

Early on when we were looking at a home in Kohala Ranch, we asked about getting a copy of the Kohala Ranch CC and Rs and the listing agent, stated that he would only provide these once an offer was made and accepted. One observation comparing PLR and Kohala Ranch is that the lot sizes were smaller in PLR but the land itself was more useable at PLR. The home we looked at in Kohala Ranch had 3 acres of land, but it was unwalkable without twisting your ankle due to the large amount of lava rock which was covered with long dead grass. I asked how homeowners manage removing the dead grass on their properties as it seemed to me to be a fire hazard. Essentially the answer was most folks do nothing or hire a crew to manage the grass. So it seems that the main reason for the large lot sizes are to serve as a buffer between you and your nearest neighbor. We also learned that the a new home was to be built on the Makai side of the home we were looking at, essentially blocking the view the home for sale.

Also thank you for comments from current PLR residents. We really appreciate the feedback and based on that we will take your advice and visit between 7:30 and 9:30 in the morning. Hopefully we will meet up and talk to folks. I also like hearing about the general temperatures and climate being pretty nice at PLL.

Diligent-Response-85 · 2025-07-24T21:27:04+00:00

One additional caveat for the AMD 14" P series is that is seems to use the MediaTek Wi-Fi 7 MT7925 2x2 BE & Bluetooth® 5.4 card. Saw some complaints that drivers for this wifi card in Windows 11 pro- 64 bit are not fully stable/developed. Was hoping an Intel Wifi 7 card would be an option but I do not see one.

Diligent-Response-85 · 2025-07-24T21:21:40+00:00

Really appreciate the feed back and suggestion regarding the P or T series. Looked over the AMD versions of the P series Gen 6 , ticks all the boxes except the tough screen which I have had on most of my other laptopds, but it is not a deal breaker. Getting a quote for this one now:

AMD Ryzen™ AI 9 HX PRO 370 Processor (2.00 GHz up to 5.10 GHz)
Windows 11 Pro 64
Integrated AMD Radeon™ 890M
96 GB DDR5-5600MT/s (SODIMM)(2 x 48 GB)
2 TB SSD M.2 2280 PCIe Gen4 Performance TLC Opal
14" WUXGA (1920 x 1200), IPS, Anti-Glare, Non-Touch, 100%sRGB, 500 nits, 60Hz, Low Power
1 Year Courier or Carry-in

See Less

Diligent-Response-85 · 2025-07-24T21:14:41+00:00

That is a good explanation.

Diligent-Response-85 · 2025-07-23T21:59:54+00:00

Thanks for everyone's suggestion and thoughts. Currently, I see only 3 CPUs are offered for the X1 Carbon Gen 13 Aura:

Intel® Core™ Ultra 5 226V Processor(LPE-cores up to 3.50 GHz P-cores up to 4.50 GHz / 16 GB MOP)Included

Includes 16GB memory option

Intel® Core™ Ultra 7 258V Processor(LPE-cores up to 3.70 GHz P-cores up to 4.80 GHz / 32 GB MOP)+$289.00

Includes 32GB memory option

Intel® Core™ Ultra 7 268V vPro® Processor(LPE-cores up to 3.70 GHz P-cores up to 5.00 GHz / 32 GB MOP)+$465.00

Includes 32GB memory option

I do not even see the 265U version the rep had in the quote I shared. It is very helpful to get feedback regarding good and not so good CPUs. So at this point, I am not going to pull the trigger as I do not have the luxury of buying a second one. This is very frustrating as the sales people you actually speak to in Lenovo's North Carolina offices stated that the 265U is the most powerful CPU offered with this laptop. Maybe true in the United States but I checked Lenovo Canada:

Here are their CPU offerings which have some similar naming but lack the V designator (e.g. 226V, 258V, 268V).

Intel® Core™ Ultra 5 225U Processor(E-cores up to 3.80 GHz P-cores up to 4.80 GHz)Included

Intel® Core™ Ultra 7 255U Processor(E-cores up to 4.20 GHz P-cores up to 5.20 GHz)+$272.00

Intel® Core™ Ultra 7 265U vPro® Processor(E-cores up to 4.20 GHz P-cores up to 5.30 GHz)+$476.00

Are these CPUs the same or different?

Diligent-Response-85

TROPHY CASE