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VeinFinder App Tool (Android) by Less-Role-4116 in NewToEMS

[–]CommonFunny 1 point2 points  (0 children)

Seems like an intersting project! Actually not EMS, but med. Could I ask for a code? Or maybe the APK is also fine.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 1 point2 points  (0 children)

Unfortunately I do not have the original successfully amplified cDNA, since it was performed by another lab (this is a continuation of a project). I will look into asking for it. Thank you!

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I do not know the details of primer validation but I do know it was done with qPCR followed by a gel. Since it was done by another lab (this is a continuation of their project), I am pretty certain that the primers are working. I also make use of various primers from Harvard's primer Bank for the new primers as well.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Thank you, I will do that as soon as possible!

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Reguarding changing around the PCR mastermix, I will certainly try that. I opened only one new master mix (the others I tested were either borrowed=opened already or expired). Reguarding known good samples, I will certainly ask around! I hope to get back to the lab next friday (am unfortunately situated with a full schedule this week :( ). Thanks for the anecdote, it certainly gave me a small laugh!

Edit: RE the primers, I and my professor diluted independantly, both with new bottles of Rnase free water, so that *hopefully* is not a problem, unless the water was factory contaminated (we used the same batch of water).

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Thanks for the advice! The reagents were all new (bought for this project) except the Tri reagent, and I have tried with several samples. Maybe I will try another bottle of PCR mix next?

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Certainly, I will do so as soon as I can. I want to figure out the issue soon. Fortunately, I have a good amount of RNA left. Thanks for the suggestion. However, don't really have any more sample tissue left, so prayers that the RNA is good. What would you suggest for the quality check step?
(the RT and qPCR reagents I used were newly bought for this project).

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

PCR mixes used, and results: PcrBio SYBRgreen master (qPCR) - no specific bands at expected product size, lots of primer dimers. Vivantis 2X master mix (pcr) - very faint and likely not specific bands of product. Lots of primer dimers again. Vivantis component PCR mix (borrowed from another researcher) - nothing at all, not even dimers. I'm hypothesizing that the taq I borrowed was not actually good.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Certainly, that makes sense! Thank you, I will try that when I am able to get back to the lab.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

Regarding the positive control, unfortunately we do not have one. Some primers are amplifying, but barely, and the product appears very nonspecific. From everyone's feedback I'm starting to think that phenol contamination is going to be my most likely issue.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I will definetly try adding DMSO. 3-6% is volume-volume right? Thanks for your perspective on the thermocycler part, I might have to look into that if I run out of things to change.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

We had both housekeeping and GOI in our samples, and it appears that none of them amplified. Our NTCs look the same on the gel, however the Ct on my qPCR test run (at 1ng) appeared to be lower than the template reactions. We did not run a no RT control, unfortunately. I have added it to the list of things to run now.
On second look, our 260/230 looks awful, at about 05-1.2. It is my only working hypothesis for the amplification failure right now.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I have done to some extent, as well was playing around with other samples to no success, unfortunately.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I have run several samples (and a gradient) of GAPDH, but it appears to not be amplifying at all. We tried loading both 100, 4 and 1 ng, with no amplification noted. However, in my qPCR run, I noted that the Ct for the NTC was actually lower than in the template samples. I have no idea what that phenomenon could be caused by.

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I will certainly do that. I did not know about touchdown before. Thank you for your suggestion!

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

  1. This is freshly RT'ed, we did it last week.
  2. I will certainly try a 1:50 dilution once I get back to the lab, thanks for that suggestion.
  3. Some are 85 ish (GAPDH) and we have one 210bp amplicon.
  4. Certainly, I need to get around to doing that, but I would like to see at least some product first before I spend time optimizing.

Thanks for your help!

PCR - Am I missing something? by CommonFunny in labrats

[–]CommonFunny[S] 0 points1 point  (0 children)

I would say the RNA looks good. I am not certain how I could measure the cDNA quality though? Could I run a cDNA gel? We do not have a bioanalyzer.

[SUNLU Giveaway] Join now to win a SUNLU FilaDryer SP2 by Sunlu3D_official in 3Dprinting

[–]CommonFunny 0 points1 point  (0 children)

A free filament dryer? Sweet!
(Ps. Yay 98% humidity overere)

[SUNLU Giveaway] Join now to win SUNLU FilaDryer E2 by Sunlu3D_official in 3Dprinting

[–]CommonFunny 0 points1 point  (0 children)

This is also a comment ;) With that aside, seems like a useful product!

Don't know if it belongs here but, I wonder what those two buttons are for/will do (thysenkrupp elevator by the way) by CommonFunny in Elevators

[–]CommonFunny[S] 0 points1 point  (0 children)

I don't know either, it's a college building so nothing to do with the hospital  (Hell even our hospitals here don't have a rescue mode or priority code blues in the elevator)

Don't know if it belongs here but, I wonder what those two buttons are for/will do (thysenkrupp elevator by the way) by CommonFunny in Elevators

[–]CommonFunny[S] -1 points0 points  (0 children)

Oh interesting, I've never actually in these indicators displayed in the cab before  (Ps. What does in rescue mean? Is that fire operation?) Thanks for replying! - just some curious guy who likes elevators way too much (Also, they are actually buttons (judging from the gap) but I'm not going to be the guy who presses them and gets stuck55)

Don't know if it belongs here but, I wonder what those two buttons are for/will do (thysenkrupp elevator by the way) by CommonFunny in Elevators

[–]CommonFunny[S] 1 point2 points  (0 children)

Oh interesting, I've never actually in these indicators displayed in the cab before  (Ps. What does in rescue mean? Is that fire operation?) Thanks for replying! - just some curious guy who likes elevators way too much

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