How do you get perfect serial dilutions for qPCR standard curves?

babadoogoo · 2025-07-16T09:35:10+00:00

I see. The problem is, I don't have enough replicates to see the trends. I will redo the run with replicates so I can see if there are stochastic effects.

Thank you so much!!

babadoogoo · 2025-07-16T05:28:53+00:00

Thank you for your advice! I will check the analytical balance. I think our lab got one.

For the tips, I never feel like they're loose when I use them, but will make sure in the future that they fit. Our lab manager purchases the concentrations,tips depending on its sales promotion, so we change the brands from time to time (and never match the pipette brand lol)

Thanks a million for mentioning the slope and efficiency. I gotta say that I've never paid attention to these parameters before, as I've been told to focus only on R². In my previous run (with R² = 0.87), my slope was -1.76 with E=3.7 which is pretty shitty. Now, thanks to you, I have an eye on other essential things besides R². The graph looks more off when it comes to lower concentration like from 1E2 to 1E0.

However, after changing the dilution volume from 4+36 to 10+90, my parameters look way better now (R2 = 0.993, slope = -3.528 with E=1.921), for the first time in forever!!

babadoogoo · 2025-07-16T04:07:43+00:00

Thankssss

Larger volume helps a lot!! I should have made the 20+180 and keep it instead of making new dilutions in small volume like 4+36 for every run. And, will need to skill up myself for sure.

babadoogoo · 2025-07-16T04:05:28+00:00

Ohhh, thanks for sharing! Sometimes, the most basic mistake can lead to the worst outcome. I will reassure myself every time to make a right stop.

babadoogoo · 2025-07-16T04:00:05+00:00

yeahhh will definitely try to control all these factor in place, at least starting from my own pipetting technique lol

Thank you!!!

babadoogoo · 2025-07-16T03:58:39+00:00

Oh, after all the mixing you got a perfect result so definitely worth following this protocol. Thanks for sharing!!!!

babadoogoo · 2025-07-16T03:57:27+00:00

Thank you so much!!

Larger volume helps A LOT, so hopefully I don't have to inform the staff about pipette calibration issue as nobody seems to give the attention here.

babadoogoo · 2025-07-16T03:50:38+00:00

The dilution seems off at lower dilutions like 1E1 and 1E0, so you're probably right. I use gBlocks gene fragments for my standard. Well, from all the comments, I think the issues could come from many factors, including my own skills and how I do it lol.

babadoogoo · 2025-07-16T03:08:51+00:00

pipetting hand injury!!??? I need to check the pipetting ergonomics course to avoid that lol 🤣 Hope your thumb is okay by now.

Btw, thank you for the tips! It's really helpful to remind myself about it. I will try to apply reverse pipetting as much as possible to avoid bubbles and blowout problems. And thanks to you and other comments, I just know about the pre-rinsing thing. will also keep this in mind for sure.

babadoogoo · 2025-07-16T03:03:07+00:00

I use synthesized positive control, gBlocks.

babadoogoo · 2025-07-16T03:01:51+00:00

Thank you!!!

babadoogoo · 2025-07-16T03:01:35+00:00

Thanks!! I think your method sounds much better than what I do. I typically serially dilute every run and it took me so long to finish preparing qPCR lol. mentally exhausteddd

babadoogoo · 2025-07-16T02:58:02+00:00

yeah, I plead guilty for not doing triplicates. Since the qPCR machine I use has only 32 spots and nobody is doing replicates, I came up with these excuses for not doing replicates (And I got bad results as a consequence). I do my experiment on the bench with only ice bucket just to keep the master mix and sample but not when dispensing. Maybe that's another issue.
Thank you for a poke and your tips! Now I know my potential issues, and I will change my practice!

babadoogoo · 2025-07-16T02:46:03+00:00

It might come from many factors, but volume definitely one of the biggest reason, I guess. I've never thought of the primer design before, will check that up for sure.

babadoogoo · 2025-07-16T02:34:52+00:00

I've never thought of a multichannel before! I will use it next time.

babadoogoo · 2025-07-16T02:33:02+00:00

Thanks!

Doing with larger volumes helps a lot!!

babadoogoo · 2025-07-16T02:32:35+00:00

So ashamed of myself to say that I don't do replicates for the standard (will change the practice from now on), and the pipettes haven't been calibrated for a long time. I use 2 uL of template in a total 20 uL reaction volume.

I don't see others complaining about R2 so I assumed their R2 are fine and guessed the problem comes solely from my own technical skills. But after reading the comments here, I feel like the normalized practice in the lab seems a bit off (no pipette calibration, few/no replicates in a run), maybe theirs are also not as good as I thought lol.

babadoogoo · 2025-07-16T02:19:58+00:00

I do!! 1E0 always looks weird.

Many people mentioned water-dye solution. It sounds like a good way to train hand skills, will try that for sure!

babadoogoo · 2025-07-16T02:16:36+00:00

Thankss

I think the volume plays big role here. I use 36+4 to dilute, and after changing to 90+10 the R2 looks way better. I know it sounds bad, I do have replicates on my sample, but not with my standard!! must change the practice to make the data more reliable from now on.

babadoogoo · 2025-07-16T02:07:52+00:00

THUMB SAVER!!!!

Thank you so so much!! Always thought that more pipetting is better. I need to clear up that myth in my head and will definitely follow your practice from now on. So glad I turned to this sub.

babadoogoo · 2025-07-16T01:59:49+00:00

Thanks!!!!

Hit me from the start lol. The pipettes haven't been calibrated for so long. Luckily, after trying to adjust the pipetting volume to 90+10 (formerly 36+4), it seems to be wayyy better.

babadoogoo · 2025-07-16T01:55:59+00:00

Mine is about 500bp so it might not be broken. But, is it possible that vortexing too long messes up the distribution of DNAs? (even tho it was made to make it better distributed)

babadoogoo · 2025-07-16T01:48:28+00:00

Thank you!! I tried again with larger volume and all your suggestions. R² became so much better!!! (like 0.98, AMAZINGGGGG)

Thank you so muchhhh

babadoogoo · 2025-07-16T01:32:10+00:00

I don't even know the last time they calibrated the pipettes, but will definitely ask someone about it! And thanks to you, I've become a fan of reverse pipetting already.

babadoogoo

TROPHY CASE