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Hummingbird2's Content

There have been 8 items by Hummingbird2 (Search limited from 25-October 19)


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#194630 Light genotyping PCR bands

Posted by Hummingbird2 on 24 August 2020 - 02:58 AM in PCR, RT-PCR and Real-Time PCR

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




#194608 Light genotyping PCR bands

Posted by Hummingbird2 on 21 August 2020 - 05:54 AM in PCR, RT-PCR and Real-Time PCR

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!!

 

Cynthia smile.png




#194601 Light genotyping PCR bands

Posted by Hummingbird2 on 20 August 2020 - 03:45 AM in PCR, RT-PCR and Real-Time PCR

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

Attached Thumbnails

  • 13h33m35s(Ethidium Bromide).jpg



#194511 PVDF Membrane Floating in Reprobe Solution . . .

Posted by Hummingbird2 on 07 August 2020 - 01:42 AM in SDS-PAGE and Western Blotting

Okay - that's what I thought you meant, but that's kind of the opposite of what you wrote previously. I got you though! I do those washes the same way I do all my other washes and everything else is always fine (even though those reagents are also made in the same DD H2O). There must be something about washing the membranes in *just* DD H2O . . . weird. I'll just do one quick wash in DD H2O before adding the reprobe in the future! Easy fix! Thank you!

 

Cynthia




#194503 PVDF Membrane Floating in Reprobe Solution . . .

Posted by Hummingbird2 on 06 August 2020 - 08:55 AM in SDS-PAGE and Western Blotting

Yes - it does start to float during the DD H2O washes after MeOH re-activation . . . 

 

Cynthia 




#194497 PVDF Membrane Floating in Reprobe Solution . . .

Posted by Hummingbird2 on 06 August 2020 - 06:41 AM in SDS-PAGE and Western Blotting

Hi, all - I use an older BioRad system for western blotting. The membranes I use are just a standard BioRad PVDF on a roll that I cut to size. The membranes work great all throughout the blotting process . . . unless I have to reprobe one . . .

After exposure I wash the membrane a couple time with TBS-T and then let dry out. Once dry, I re-activate with some methanol for a minute or so. Then I wash a couple times with DD H2O and put it in the 1X reprobe solution (5X solution from G Biosciences, diluted to 1X with DD H2O). The problem is, the membranes always float on top of the reprobe solution! No matter how much I poke them down or swish things around, they rise to the top again! 

I know PVDF is hydrophobic, but I never have any issues with any of the other reagents (TBS-T, antibodies, milk, etc) which are also made in DD H2O - PLUS I asked a colleague who reprobes all the time about this and she said she's never had issues with membranes refusing to submerge into reprobe solution (and she uses the same exact brand I do and dilutes it in the same exact DD H2O that I do)!

 

So what is up!? Any suggestions??

 

Cynthia

 




#194324 Can't remove Ponceau staining!

Posted by Hummingbird2 on 18 May 2020 - 04:03 AM in SDS-PAGE and Western Blotting

Well, I washed the membranes really well with TBS-T on Friday and let them dry completely over the weekend. I just put them back in methanol and like 99% of the Ponceau came out! It took a couple minutes, but it came out! Weirdest thing ever . . . so, problem solved - but if anyone has any insight as to why this happened, I'd still be interested! Tx! 

 

Cynthia :) 




#194323 Can't remove Ponceau staining!

Posted by Hummingbird2 on 18 May 2020 - 02:59 AM in SDS-PAGE and Western Blotting

Hi, all - First post! biggrin.png

So, 
I have always stained my PVDF WB membranes with Ponceau to get a background image, then removed the Ponceau stain by adding a bit of 100% methanol and swirling around on the membrane for a minute or so before rinsing and blocking, etc. I ran 4 WB's on Friday, added the Ponceau, and took the pic . . . but methanol won't remove the Ponceau stain! I mean, it made the red stain a little lighter, but usually 100% methanol takes the Ponceau completely off in a minute or so! I left the membranes in there for like 10 minutes - only the tiniest bit lighter!! I even dumped the methanol and tried another lab's methanol for several minutes - nothing! I have literally done hundreds of WB's and the methanol has always taken the Ponceau off *completely* and very rapidly! Any clue why it won't take the Ponceau off now!? I haven't changed anything - same membranes, same methanol, same Ponceau, same WB procedure . . . what is going on?? Thanks! . . .

Cynthia :)





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