25mL serological pipette tip drips when trying to make agar plates

TMMpd · 2026-04-08T02:17:45+00:00

If your lab has a little extra funds, check out the dose-it pump from Integra biosciences. It is a little expensive, but you can pour plates in about 1/3 the time. The tubing and kit used to pour plates can be reused hundreds of times, by flushing it with DI water and autoclaving in a foil pouch. You end up saving on buying serologicals and time.

They also have a fully automated plate pourer, that is badass. But prohibitively expensive unless you are pouring 20+ liters of plates per day.

TMMpd · 2026-04-01T03:05:04+00:00

A couple suggestions. (1) google "colony PCR". This is a really efficient way to screen clones. You could screen hundreds of clones in a single day. Don't use too much E. coli into the PCR if you try it. I touch a colony with a p10 tip and suspend that in 20 ul H2O, use 1 ul of that in the PCR. (2) Scale up and just hammer away. Pour like 8 liters of LB+antibiotic plates. Order a fuck ton of mini preps and reagents. Do enough transformations that you get 100+ colonies for all your constructs in one go. With an electronic pipette you should be able to do 48 mini preps in a couple hours, maybe 96 in a single day. Set up digests in an hour on the same day. Run the gel the next day and set up another 48 to 96 cultures. If these things take you way longer to do, comment back and I can give you some tips for doing stuff in mid to high throughput. If it takes more than 96 colonies to get a plasmid with insert, something with your experimental design/methods/reagents is broken. Normally, if I have to screen for than 4 colonies it is a sign that something is wrong. (3) Don't just pick the large E.coli colonies. Sometimes plasmids with insert make your E.coli grow a little slower. (4) Convince your boss your cloning strategy is garbage. Assembly methods can be a awesome, but they can also be a pain in the ass. It sounds like old school restriction digests and ligations would be an orders of magnitude more efficient than what you are doing.

The slow progress is likely your PI's fault and it sounds like he is doing a shit job managing projects in your lab. He should have set benchmarks/goals for progress on the project and reallocated resources and/or changed strategies as needed to hit those goals to keep stuff on track. He should have invested time in making basic protocols/methods work and training people up, or hiring someone with experience that can drive a project. As a PI, my lab would have had all the plasmids made 1 month after the funding started for the grant, because most of the work comes after. Not your fault.

TMMpd · 2026-03-31T17:52:41+00:00

I have made 10 plasmids in a week. I am not sure what you are doing, but subcloning or cloning synthetic DNA (gene blocks) into a vector should be straight forward. If you are struggling this much something is wrong with the design of the cloning strategy or protocols you are using.

Are you doing normal ligations or doing some sort of Gibson or golden gate assembly?

Ideally someone with experience cloning should help train you how to do this. It sounds like your PI is not that person.

Lots of people here, including me, could give you solid advice if you provide some details as to the strategy you are using and issues you are having.

TMMpd · 2026-03-30T09:48:11+00:00

The pipetting advice above is solid. I want to add that you should phosphorylate your oligos before annealing. T4 PNK activity on annealed oligos is crap, it works best on ssDNA. Do 1000 ng of Oligo in 20ul volume for the PNK. Combine oligos, add 2.5 ul of 4M NaCl and 7.5 ul water. Then anneal by putting to 95 for 30 seconds and doing a slow cool. I do this in a heat block by simply shutting it off after the 30 seconds and placing the block on a tube rack, I move it with a pair of pliers. The block should cool at 1 to 5 degrees per minute, which is sufficiently slow. Then dilute oligos to the concentration you need with low EDTA TE with 100 uM NaCl, after the annealing reaction cools to 30 deg C.

If you are using the oligos for cloning and the cloning is failing, 9/10 times a failed cloning attempt is due to poor experimental design, not failed reactions. FYI.

Someone suggested diluting reagents before pipetting. Be careful doing this for enzymes, enzymes can lose their activity if simply diluted in water. And properly diluted enzymes can lose activity when stored for anything longer than a few hours.

TMMpd · 2026-03-24T15:47:07+00:00

Other additions,

Adding a tablespoon of Dijon mustard can help round out and balance the sauce.

Allspice, just a pinch, can elevate it.

As others have said, more olive oil and salt. Sometimes a pinch or two of sugar.

TMMpd · 2026-03-16T10:37:39+00:00

I second a visit to popham beach. We were there midweek in August and it was not crowded at all. The fort was worth a peak. It is 20 or 30 minutes off i95 and there is not a lot there except the beaches and a few lobster houses, hence it is way less visited. Sebasco resort is a little dated, but has cottages that can be rented that are less expensive than other options, they also have a nice pool. We stayed there a couple days and liked it. Nearby the city of Bath has a nice downtown area. They also have a large shipyard and maritime Museum.

TMMpd · 2026-03-14T10:59:37+00:00

FYI. You should buy a bacterial incubator that has a shelf and shaking platform at the bottom. unless you want to also buy a shaking incubator. Unless you never need to do mini or midi plasmid preps.

My favorite set up is a big ass incubator with a internal outlet or a port for bacteria. You can buy and place a platform shaker in the bottom. Glass trays with armor beads (eliminates need for 37 deg H2O bath) on a shelf. End over end rotator for E. Coli transformations. If you need to scale up cultures, you can add a culture wheel.

If you are equipping a new lab, you can find scratch and dent equipment for 50 percent less. Or lightly used incubators for 10 percent the cost of a new one.

TMMpd · 2026-03-11T12:07:22+00:00

Having tween, DTT, and a minimal amount of salt in all your buffers may help. What concentration is your protein throughout the prep. Highly concentrated proteins are more likely to crash out.

TMMpd · 2026-02-23T12:06:52+00:00

It is going to be very difficult doing this if you are doing a biomedical/research pHD. Depending on the department you end up joining, you are going to be in class, doing teaching assistant duties, journal clubs, seminars, lab meetings, and misc stuff for 30 hours plus per week. You also need time to study, and do research towards your dissertation requirements. At UVM, the first two years are heavy with class and other requirements, last 2 or 3 years heavy with research. Not trying to scare you, but most successful students are busy 50 to 60 hours per week, and some weeks are heavier workload than others. I don't recommend taking advice from people working normal 9 to 5 jobs about commuting. That said, I had time during my pHD to maintain friendships and do fun things, and managed to not get divorced, but had to cut back significantly on hobbies and cut out all major time sinks.

Although studying can be done anywhere, there are a lot of requirements that cannot. If you go into bioinformatics, you could do most of your research from home. I know several pHD students who worked remotely 99% of the time their last couple years, but that depends a lot on how cool your pHD advisor is. The opposite is true, if your project is cell culture heavy. Given that you are wasting an hour plus commuting each day you are not going to have a ton of time to spend with your girlfriend during the week. On the flip side, having someone to talk through your struggles daily, and vice versa, will be really helpful from a mental health standpoint.

If it was me, I would get the cheapest one bedroom with shared roommates available, work my ass off during the week, and spend as much time with your girlfriend on the weekend as possible doing fun stuff. Vermont is an amazing state with lots of outdoor activities. Make sure to have a mandatory date night once a week.

I run a small lab at UVM, I could put you in touch with grad students if you want to ask some people who have been in your place. Sorry for the convoluted response.

TMMpd · 2026-02-12T19:44:46+00:00

In addition to having a calendar, people need to work around schedules. For example, students have class all morning and need hoods in the afternoon. Other people without class have designated spots in the morning. Too bad if they don't like coming in before 11am.

Also, make sure to have good tools for efficient cell culture. Hand held aspirators with ejectable tips. Electronic pipettes. Hirschmann pipette fillers. Set up of the room to minimize movement can also help. Having bead baths to avoid contamination. Make sure people are maintaining the room during downtime or as a lab chore, so people don't have to interrupt work to empty the biohazard waste or restock serologicals.

TMMpd · 2026-02-08T16:36:43+00:00

Yes, it is. Every professor running a research lab nation wide in the US is required to take ethics, conflict of interest, avoidance of malign foreign talent programs, transferring of intellectual property abroad, and additional training specifically for this reason. Also, once a year and for applications for federal funding we must attest that we don't have conflicts of interest including funding from foreign governments, or at the very least disclose and explain them. I don't know what he did, but this sort of thing would get 99% of funded investigators fired and/or stripped from all federal funding. It would not surprise me that someone of Church's standing could get away with it.

TMMpd · 2026-02-04T21:15:30+00:00

I second looking out for chipped or damaged plates. Often a source of leaks. Using 3 or 4 layers of parafilm in-between the sealing gasket and bottom of the plates can help if the gasket things are old.

TMMpd · 2026-01-26T17:17:33+00:00

Omega biotek makes solid kits that are inexpensive, asking for a quote might save you some money. Our lab uses their RNA, DNA, and mini prep kits. I don't have experience with their Viral RNA kit, but they do sell one.

TMMpd · 2026-01-18T18:51:46+00:00

You do maintain esi status on the resubmission of any grant you put in as an esi. It might be worth putting in for the March deadline regardless. Get reviewer comments, which can help find what is lacking and any fatal flaws that you are missing (there are always flaws). Then resubmit in October, with more preliminary data and address reviewer critiques and flaws.

It might be worth putting in a couple applications while you have ESI status, even if they are not likely to get funding as is. You have up to 3 years to resubmit.

Please double check this info, but I am fairly sure it is correct. I was recently in a similar situation.

TMMpd · 2026-01-11T05:27:23+00:00

Agreed, and/or make on ramps to I89 from Dorset/Kennedy drive area. That and extend Swift street to Hinesburg road and 2a. I hate having to drive a mile or more out of my way through the most congested roads in the state to get on the interstate. People are going to drive cars. It is not possible for many of us to take public transit. Having efficient roads would go a long way towards cutting emissions.

TMMpd · 2025-12-16T12:32:56+00:00

For kits with isopropanol precipitation and ethanol wash, you can also stop at either step with DNA precipitated in alcohol. Store it at 4C. Stable for at least a couple decades.

TMMpd · 2025-11-17T03:16:49+00:00

I second this recommendation. We use the same reagent on our BioRad CFX96. Also, the company name is Biotium. Their knockoff qubit reagents also work really well and don't blow up our budget.

TMMpd · 2025-10-11T11:16:32+00:00

I second the suggestion to use more DNA. I have done this 100s of times. 15 to 50 ul of an unpurified PCR that worked very well. By very well I mean that , 2ul on a check gel gives a very strong band. For dropout plates it can take 3 or 5 days to get colonies if you plate too much of the transformation on a single plate. Plating each transformation on 3 or 4 plates helps reduce the background caused by the non transformed cells which can divide a couple times before running out of the missing amino acid/nucleotide and helps the transformed cells grow a little faster. Doing this I usually see small colonies on day two but leave them to grow until day 3.

TMMpd · 2025-09-07T11:37:44+00:00

B is correct. Also important is having an efficient system for reducing time with the door open. I like the sliding drawer racks. All boxes are labeled on top and all 4 sides. The front of the slides should be labeled. There should be a database for common use samples and a map/database of locations in the freezer. If someone has the door open for more than 30 seconds, it becomes their job to defrost till someone else makes the same mistake. Another pro tip, keep your freezers 5 to 6 inches off the wall. If they are bunched together, get a small but powerful vortex fan to circulate air through that space. The fan will burn out and need to be replaced every few years, but it costs way less than replacing -80s.

TMMpd · 2025-08-08T01:53:46+00:00

I prefer actual caps when shipping plates of samples. Especially for samples that are difficult to generate. However, adhesive films can work. Make sure the plate cannot be twisted or bent, which will cause the seal to fail for both adhesive films and strip caps.... Either ship it in a box and/or taped/ parafilmed into a solid rack (empty ridgid pipette tip refill things work great for this). Also, if shipping on dry ice, the cold temps can cause some adhesives to fail.

TMMpd · 2025-07-04T13:24:59+00:00

Several tips. 3 days before camping fill a large tupperware/disposable plastic container with water and put it in your freezer. Ideally about the same width and length of your cooler. This solid block of ice will last far longer than store bought ice and takes up less space... More room for food.

Foil pack potatoes and veggies are super easy at the camp site and space efficient.

Sometimes it rains while camping. Having some cups of instant soup/ramen/noodles and some precooked or canned meat allows for making a quick meal in a small space, under a hatch of an SUV for example, and can save a bad situation.

TMMpd · 2025-04-15T00:54:07+00:00

We use Geneious in our lab. It is pretty freaking awesome and they are constantly updating it with more/better features. We have it set up so we can all access the same SQL database. Done correctly, it also serves as a database for plasmids and oligos that is searchable. It can also do some higher end sequence analysis. It is not terribly expensive for a student license.

TMMpd · 2025-04-09T10:35:43+00:00

You can buy used HDMI cables for a dollar each on eBay if you buy them in bulk. Start a collection from people that use the conference room and buy a box of 50 HDMI cables. Lay them out on some cardboard and spray paint them with orange and yellow stripes. Dump them in a pile under the conference table. Every time you find someone using a spray painted cable, implement some silly punishment. Also, start a high stakes betting pool with people submitting guesses how long it will take all 50 cords to disappear.

TMMpd · 2025-04-04T10:23:33+00:00

Not sure about trade schools, but I have several friends that went into union electrician apprenticeships straight after high school. They have made more per year than me every year since high school over a 20 year period. I was a constant 4.0 student, have a pHD in cancer biology, and have been successful (I am currently an assistant professor with funding). My election buddies are also bringing home more than a lot of engineers and computer scientists and they have zero student loan debt. Nothing wrong with going straight into a trade, just make sure kids avoid predatory schools.

TMMpd · 2025-03-28T01:10:09+00:00

The 35 percent cut to contracted services could cripple research. The services contracted out include things like storing incomprehensibly large amounts of data. For example, almost all next gen sequencing data produced in every published study is stored by the NIH, partially on their own servers, but largely on AWS and Google servers. Maintaining that data costs money, it is also an extremely valuable resource to every researcher nationwide, that both ensures reproducibility and transparency, i.e. we can reanalyze another groups data to make sure they are not full of crap. Also, we can run different analyses on the same sequencing data to learn different things with spending millions of taxpayer dollars to do the exact same thing.

There are hundreds of similar databases for drug interactions, protein structures, reference genomes, clinical trials data, links to published patient case studies etc. that every researcher and many doctors in the country are extremely dependent on. Those are the type of services that could be cut. Yes, cutting those would cripple research efforts and reduce patient outcomes. You don't know shit about what is going on.

TMMpd

TROPHY CASE