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Light genotyping PCR bands


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#1 Hummingbird2

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Posted 20 August 2020 - 03:45 AM

Hi, all! I've been struggling with this forever . . . I have 2 different genes that I do PCR genotyping for and there are always super bright obviously-positive bands and then bands of varying degrees of brightness. I attached an example image where you can see about half the samples are very bright and obviously positive, 4 are pretty obviously negative, and 2 are kind of ambiguous (sample 2 and 6). Sample 2 is brighter than 6, but neither is remotely as bright as sample 1, for example. So are these pos or neg??

I ran a known pos (which was very bright) and a known neg (which was about the same brightness as mystery sample 6) and a water sample (which was totally blank). But sometimes my known neg is totally blank!

 

So do I base my pos/neg decision on the brightness of the known neg sample each time I run? Like, if I run a known neg and it is completely blank, then do I count any sample in that run that has any hint of a band as pos? And if I run a known neg and it has a faint band, then do I count any sample brighter than the known neg as a pos, and any sample lighter than the known neg as a neg!?

And it's all totally random as to whether the known neg has a faint band or not. And I've changed out every single reagent a million times and it usually doesn't help. I've also tried diluting the DNA and it usually doesn't help. And it does this for both of my genotyping protocols (a K5rtTA and a GFP). 

 

I'm using an overnight homemade DNA isol buffer shaking at 55C and then 10min at 95C in the morning. 

 

Any help would be appreciated!!

 

Cynthia smile.png

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Edited by Hummingbird2, 20 August 2020 - 03:47 AM.


#2 bob1

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Posted 20 August 2020 - 01:14 PM

Short answer - Generally a known negative should be negative - produce no band at all under any conditions. The fact that you are seeing a band in your known negative under your conditions here indicates that either you have some form of contamination of your sample or a poorly designed primer pair.

 

A quick test would be to re-run the PCR using a completely new aliquot of your known negative and completely new PCR reagents. Set up a few independent reactions (~10 - make them up individually, not from a master-mix of reagents) and see if any of them come up weakly positive. If they do then you have a problem with the PCR itself or perhaps the "negative" isn't actually negative. In this instance it may help to sequence the band from your known negative to see if it is giving a product that matches the positives or a non-specific band.



#3 Hummingbird2

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Posted 21 August 2020 - 05:54 AM

Yeah - I'm gonna take some plain old FVB ear punches and digest them and use them as absolute 100% negatives . . . along with all new reagents - literally for the 100th time. Do you do your DNA and PCR prep in a hood? I just do it on the bench top wearing gloves. Do you think I'm just getting constant contamination in my samples/reagents? How do you clean your ear punch between mice? Should I be using some sort of DNA Away spray on them between each mouse? That seems like it would be a major contamination factor, but nobody I know uses anything on their punch between mice . . . Let me know! Thank you!!

 

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#4 bob1

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Posted 21 August 2020 - 03:25 PM

It is likely that the contamination is coming from the lab somewhere, basically you re-amplifying already produced PCR products. PCR contamination is a huge issue and very hard to get rid of as it can float around in the air as aerosols (from people opening tubes), it can be on and inside pipettes, on your bench, on the fridge/freezer handles etc. I would thoroughly decontaminate your pipettes as a starting point - bleach works well for this, and use filter-tips, especially for adding template to your reactions.

 

I would doubt that contamination is coming from the clippers, but it certainly pays to at least give them a wipe with ethanol or something between mice. Generally the amount of DNA from an extraction would swamp the stuff coming from small amounts of contamination on the clippers.

 

There are a few ways of getting around this. The most common way is to have dedicated pipettes for use only with PCR products (e.g. loading gels only). Other pipettes are used to set up the reactions, and, if you want to be extra careful, a third set for adding the extracted DNA template. Another additional step is to set up the reactions (without DNA template) in another lab and with pipettes used only for that purpose, where they don't use the same PCRs as you, then take your tubes back to your lab, add the DNA, seal and run the PCR reaction. Make sure that you have a dedicated gel loading area away from your bench.

 

You can also get laminar-flow hoods with UV lights for setting up reactions. It pays to have one for reactions only (no template at all) and one for adding template only, each with dedicated pipettes.



#5 Hummingbird2

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Posted 24 August 2020 - 02:58 AM

Hello again! I do already use filter tips and I have a set of genotyping-only pipettes - not a set for each step of the process, though. And I have a multi-channel that I use just for gel loading. We've considered getting a bench top hood with UV, we should probably proceed with that. No way the boss would 'okay' two of them with their own sets of pipettes, though . . . 

 

How should I bleach the pipettes? Just the outside? Or take the shaft apart and wipe down the inner parts? Or soak?

 

Thanks so much!

 

Cynthia smile.png






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