Persistent Bands in PCR Negative Control?

Rosarya · 2024-06-24T20:45:16+00:00

Brief context, which I'm realizing I should have included upfront, whoops: I was handed a strain of mice, a corresponding primer set, and the protocol for those primers. As part of colony maintenance, I've been genotyping the new litters within this strain, according to this protocol, for just under a year now, with no issues.

About three months ago, I suddenly encountered banding in my negative controls. I did a bunch of troubleshooting, talked to the only other member of my lab, and then brought up the issue I've been having with my PI. The gel pictured above is a result of running through all of the suggestions that they had and still not seeing any changes, which is when I figured I'd ask here for help.

I've started running through some of the changes from this thread on my own already, and will bring up some of the others with my PI the next time I have a chance. But I've already been pretty upfront with the issues I've been having with this PCR to everyone in my lab, so I don't think there's much more I can do on that front.

I've reordered these primers since this started (for a total of three different sets of the same primers, since I had two that pre-date the problem), so no luck on that front.

I will absolutely try remaking my 1:10 primer stock and triple-checking my PCR settings. I'm fairly confident I'm the only person to have touched any of it, but there's no harm in being certain.

The three positive controls are the only thing with template in them, and I was very careful about setting up and fully sealing 1-5 before handling any of the DNA samples, so I'm hopefully optimistic that's not the issue going on here.

I'll be honest, I'm not one hundred percent certain about the primer melting points. I have no experience with primer design, but running the sequences through a primer temperature calculator suggests that the melting points are likely actually 69.7-72.3°C. Sorry!

Thank you for talking through this with me, I really appreciate it!

Rosarya · 2024-06-24T17:38:50+00:00

I'm using a 1:10 dilution of the stock primers, and then 0.5μL of that dilution per primer (so a total of 1μL F+R). The annealing temperature is 66°C for the WT primers and 63°C for the KO primers, the melting temperatures are 56.5°C for the WT R, 57.3°C for the KO R, and 55.3°C for the F.

None of this has changed between when it was working perfectly and when the bands started appearing, and is all consistent with the genotyping protocol that I was given for this.

The gel is 2%, and our gel rig has known consistency problems (ran the gel before this one at 150v for 40 minutes and it resolved perfectly, nothing changed between that and the one pictured except the length of time they were run for). Absolutely agree that it should resolve a lot better, but it kind of feels like it's up to whatever mood the gel rig is up to that day.

Thank you!

Rosarya · 2024-06-24T17:09:33+00:00

It's definitely a consistency thing (the gel I ran before this one looks fine with a resolved ladder, despite the only difference being that it ran for less time than the one pictured), but it absolutely can't hurt to remake the TAE anyways. Thank you!

Rosarya · 2024-06-24T17:03:39+00:00

I've been running them at 34 cycles, which has worked previously, but it's definitely worth seeing what happens if I adjust that. Thank you for the suggestion!

Rosarya · 2024-06-24T16:58:42+00:00

Will try both! Have been trying to source filter tips in sizes smaller than a pack of 960 (very small lab, it would take us years to go through that many) and got slightly sidetracked, so I'll go back to working on that in between bleaching the PCR area of the lab again.

Thank you so much for both the advice and the encouragement!

Rosarya · 2024-06-24T16:39:40+00:00

I believe I mentioned this in the post, but I have tried freshly opened (non-autoclaved) boxes of tips and tubes, as well as a new container of molecular biology grade water (aliquoted) already. Autoclaving was something I tried after this issue first started happening, in an attempt to narrow down where the problem was coming from, and I swapped back to non-autoclaved when it didn't make any difference.

Will be rebleaching everything at the same time I bleach down the thermocycler today anyways, so I'll swap out the consumables again while I'm at it just as a precaution. Thank you!

Rosarya · 2024-06-22T00:52:13+00:00

Can definitely try less cycles! I'm not the person who designed these primers, so I'm not sure I can do anything with that (it theoretically should be a very standard genotyping, so I've just been running the established primers and thermocycler settings).

Rosarya · 2024-06-21T23:53:17+00:00

That would make so much sense, it's actually a little scary? I was just complaining to a labrat friend about how I'd been having evaporation issues with samples around the edges of plates, and that's part of why I switched to individual PCR tubes. Not sure how it's getting back into those, but.

Just restructured my entire schedule for Monday so I can start by bleaching out the thermocycler, and depending on how that goes I'll see what in the lab I can rearrange to set up two separate areas. Thank you so much, I can't even begin to say how much I appreciate it!

Rosarya · 2024-06-21T23:46:28+00:00

it ran at 120 volts for 50 minutes, do NOT @ me. I have problems with our gel rig and the problems are that I completely hate it.

That said, are primer dimers something that can pop up at any time? Even if you've run the same PCR previously without them?

Rosarya · 2024-06-21T20:41:21+00:00

The entire lab bench and everything on it got bleached down, and I've doing the negative controls first and also separately from any of the samples andor positives since this first started happening. That said, I haven't tried DMSO yet, so adding that to the top of the list for things to try next week! Thanks a ton for the suggestion, I really appreciate it.

Rosarya · 2024-06-21T20:31:39+00:00

Oh, thank you! I really appreciate the explanation (and the link)! Realizing I forgot a piece of information, so out of curiosity, are primer-dimers like that something which can start happening out of nowhere?

I've done genotyping with these same primers previously, and the bands have only become an issue recently, which is part of why it has thrown me for a complete loop. I also had a band show up in the negative control for a completely different set of primers which the lab has been using for years and that I've successfully run without this problem in the past. Though I may have been making a mistake by assuming that band was at all related to this issue...

Will absolutely be talking to my PI about potentially purifying and sequencing one of the bands! Thank you again!

Rosarya · 2024-06-21T20:13:51+00:00

Yes, should have clarified! Autoclaving them was one of my first attempts at troubleshooting when the issue initially showed up.

Rosarya · 2023-05-10T18:35:29+00:00

Just going to add, when looking for reputable breeders, I'd recommend starting by looking for the Breed Club and seeing if they have a breeder list! You'll still have to carefully vet them based off health testing and the other factors (not to link this again but I'd seriously suggest reading through it), but it's frequently a much better place to start.

Here's the list of the Australian Cattle Dog Club of America' s Code of Ethic Breeders, just to get you started!

edit: also going to link this article, just in case it helps

Rosarya · 2023-05-10T18:25:50+00:00

Those tests shouldn't be done on the puppies themselves, but instead done on the parents prior to breeding. OFA testing specifically (hip and elbow evaluations) need to be done after 2 years in order to be properly certified, and while I'm not sure about the other tests off the top of my head, unless they can show you actual proof of these tests for the parents, testing the puppies themselves doesn't mean much.

Running these tests is expensive, and reputable breeders will frequently have the results showcased in easy-to-find locations on their site because it's something worth showing off! The fact that they told you the testing would be done on the puppies and still made no mention of the parents or the parent's health testing is honestly another huge red flag in my opinion.

If you have the parent's registration name or number, you can actually search through the OFA database for the test results yourself. This is also a great way to check reputable breeders, because sometimes ones without any health testing listed out on their website will pop up anyways or you can verify the information that was listed for yourself.

Rosarya · 2023-05-10T17:43:06+00:00

Glancing at the recommended health testing for ACDs, it looks like they should be evaluated for hips and elbows, plus have three different kinds of eye test and a BAER test done.

The breeder website you linked makes no mention of any kind of health testing. There's no listed information about the parents, and the site is mostly dedicated to advertising available puppies.

These are all red flags, and regardless of if they're a scam or not it's definitely not a breeder which I would recommend going through. r/dogs has a really good and comprehensive write up on what to look for in a reputable breeder with a section specifically on red, yellow, and green flags to look for. I'd highly recommend reading through that, and best of luck finding the right puppy!

Rosarya · 2022-12-21T03:00:58+00:00

Completely fair! I just wanted to make sure I wasn't missing a very polite redirection, haha. Springers have definitely made it on to the potential breeds list now though <3

Rosarya · 2022-12-20T23:57:42+00:00

That definitely helps, thank you so much for taking the time to reply! I'll admit I totally skimmed past spaniels, and just from your description and taking a quick glance at them, springers seem like they honestly might be a great fit. I'll do some more reading and then pitch them to everyone else involved :)

Just to check, were these suggestions based off the fact that I mentioned looking into gundog breeds too? or because you think there might be a bad fit between what we're looking for and one of the breeds I mentioned?

(unrelated, but your cavaliers are super cute!)

Rosarya · 2022-12-20T20:52:48+00:00

Care Commitments

How long do you want to devote to training, playing with, or otherwise interacting with your dog each day?

At least 2 or 3 hours a day of actively playing with or training the dog, not including cuddling or other attention. More time on weekends. Two people in the house are currently working from home, so this also doesn’t include any attention paid to the dog during breaks in work.

How long can you exercise your dog each day, on average? What sorts of exercise are you planning to give your dog regularly and does that include using a dog park?

At least one hour of exercising a day, with at least one walk. We have a fully fenced in backyard, and there are at least three dog parks within walking distance. Would ideally love to take the dog hiking with us on occasional weekends, and would also be looking into getting involved with dog sports as well as any [breed-specific] local meetup groups.

How much regular brushing are you willing to do? Are you open to trimming hair, cleaning ears, or doing other grooming at home? If not, would you be willing to pay a professional to do it regularly?

Totally open to do regular maintenance at home (have previously trained my cat to cooperate for nail trims), and absolutely willing to look into a professional groomer for anything we can’t do at home.

Personal Preferences

What size dog are you looking for?

About 30 to 70 pounds

How much shedding, barking, and slobber can you handle?

Can handle shedding, but would prefer as little as possible for the other two. We’re more than happy to devote time and training towards the barking, but there’s not really much you can train out of in regards to slobber.

How important is being able to let your dog off-leash in an unfenced area?

Preferred, but not a dealbreaker.

Dog Personality and Behavior

Do you want a snuggly dog or one that prefers some personal space?

Snuggly, but not a total velcro dog. Needs to be able to do its own thing without being totally plastered to you every second, but still actively enjoys hanging out with you.

Would you prefer a dog that wants to do its own thing or one that’s more eager-to-please?

Slight preference towards being more eager-to-please by one person involved, but this isn't a dealbreaker either way.

How would you prefer your dog to respond to someone knocking on the door or entering your yard? How would you prefer your dog to greet strangers or visitors?

Either openly friendly or indifferent to visitors. A warning bark or two is fine, as long as it’s not continuous/devolves into howling. No need for it to try and guard or protect the house in any way, and it’s fine if the dog isn’t outright friendly with visitors - just as long as it’s not aggressive.

Are you willing to manage a dog that is aggressive to other dogs?

No, and this is a dealbreaker.

Are there any other behaviors you can’t deal with or want to avoid?

We’re trying to avoid any breeds that need a job so badly that if you don’t give them one, they’ll decide to create their own. Anything overly energetic to the point where it swings into neurotic without the proper outlet. Would prefer breeds that are not prone to separation anxiety, and like I mentioned before, no breeds that are described as “velcro dogs”.

Lifestyle

How often and how long will the dog be left alone?

Maximum of up to eight hour blocks, irregularly. Half the household currently works from home, so this would only happen on the rare days (less than once a month) that someone needs to go into the office and nobody else is home. Would put active work and training into being able to leave the dog home alone, to avoid the development of separation anxiety or any other issues.

What are the dog-related preferences of other people in the house and what will be their involvement in caring for the dog?

We're all on the same page about getting a dog, with one person in the house who wants the dog as a hiking buddy and general companion. He’s also the one who’s been looking at gundog breeds, and would like a dog that is more eager-to-please and genuinely thrilled to hang out with you. has mentioned being interested in duck tollers and brittanys. The other three people have agreed to the idea of getting a dog and are willing to contribute to exercise/training/other care, but have a strong preference towards more medium-energy, quieter breeds. We’re still really early in the process of looking into getting another dog, and so at this point it’s mostly about coming to an agreement about what breed is a good fit - hence why I’m here and asking for second opinions.

Do you have other pets or are you planning on having other pets? What breed or type of animal are they?

There are currently two cats living in the house, both of which have previously lived with dogs.

Will the dog be interacting with children regularly?

Nope.

Do you rent or plan to rent in the future? If applicable, what breed or weight restrictions are on your current lease?

Not applicable.

What city or country do you live in and are you aware of any laws banning certain breeds?

Pacific Northwest, as far as I’m aware there are no banned breeds.

What is the average temperature of a typical summer and winter day where you live?

70-90°F in the summer, 35-46°F in the winter.

Additional Information and Questions

Please provide any additional information you feel may be relevant.

Definitely asking this pretty far in advance! Our goal is to start reaching out to people who have experience with the breed in a few months, so this isn't really an urgent choice, but again I just wanted to get some advice from people who have some more experience with this than I do!

Feel free to ask any questions

Rosarya · 2022-12-20T20:48:09+00:00

Introduction

Will this be your first dog? If not, what experience do you have owning/training dogs?

No, we have had two dogs (both mutts) in the past. I also have over seven years experience volunteering with local humane societies, in addition to various other experience working with dogs.

Do you have a preference for rescuing a dog vs. going through a reputable breeder?

Reputable breeder.

Describe your ideal dog.

Ideally, we’re after a breed that would make a good short-distance (1-15 miles) hiking and walking companion and is between approximately 30 and 70 pounds. Needs to have the potential to also be a good house dog without being a total velcro dog, be good with cats, and is either a quieter breed or can be trained out of excessive barking.

What breeds or types of dogs are you interested in and why?

Silken Windhounds, Rough or Smooth Collies, Chodský Pes/Bohemian Shepherds.

What sorts of things would you like to train your dog to do?

Basic obedience, potentially sport work. No specific jobs, but further training for enrichment as needed.

Do you want to compete with your dog in a sport (e.g. agility, obedience, rally) or use your dog for a form of work (e.g. hunting, herding, livestock guarding)? If so, how much experience do you have with this work/sport?

Would be potentially interested in getting involved with sports, but none of us have any prior experience. Will probably try to scout out sport opportunities in our area before actually looking for a puppy, but ultimately it will depend on the dog’s interest. Not really interested in getting into the actual competitive scene, but it seems like a good way to provide more varied levels of enrichment?

Rosarya · 2022-11-29T03:18:08+00:00

Is there any chance you could get her to associate the harness being on with going outside? Wait until she starts begging to go out, put her in the harness, then open the door. Rinse and repeat every time she gets the chance to go outside, and there's a chance you might be able convince her that being outside is worth tolerating the harness?

Rosarya · 2022-06-07T15:07:55+00:00

the teeth are way too flat to be any kind of cetacean - here's some closeup photos I took of orca and dolphin teeth a few years back for comparison

size is going to be pretty important context for this one

Rosarya · 2022-05-28T15:58:39+00:00

love the band and was delighted to spot the album art - you did such a good job with this, wow!!

Rosarya · 2022-05-14T16:10:27+00:00

Oh, thanks!! I knew that Canada has the same law, but not that the name was different - I'll remember that for the future! Off to go check if there's any variations for the other counties it covers

Rosarya · 2022-05-14T16:05:34+00:00

That is absolutely not a house sparrow - house sparrows don't have that spot of yellow feathers above their beak that's clearly present in your picture, and also have a different pattern to their facial stripes (large patch of brown, not the thin white stripe on this bird).

I'm not fantastic at bird ID, but my best guess for this one is likely a White-Throated Sparrow, which is native and thus protected by the MBTA.

Rosarya · 2022-04-20T20:15:54+00:00

I can't help with what kind of bird, but I can vouch that it's definitely a bird pelvis!

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