Why are my genotyping PCR bands blurry? - (Apr/27/2021 )

Hi,

I am having some problems with my genotyping PCR as the bands come out blurry. I thought this was due to either overloading or high voltage/temperature, but I am loading only a small part of my reactions and I am using chilled buffer. Also, the weak bands look more blurry than the strong bands and the ladder looks fine. What can be the issue? I have previously gotten nice bands with the same primers and I have not changed anything in my protocol apart from diluting new working stocks of primers from the "stock stock" (and maybe also a new batch of 25x TAE).

Here are some experimental conditions:
DNA was extracted from tail biopsies by incubating in 300ul 50mM NaOH under heating, then 25ul 1M TRIS pH8 was added and debris pelleted.
2 primer pairs, the products are ~250bp and ~700bp
The enzyme is DreamTaq, betaine is included in the reaction
In this run I took 2 samples and ran PCR on DNA that was undiluted or diluted 1:2, 1:4, 1:8 or 1:16 (the last well is my water control)
1.5% agarose in 1x TAE-buffer, the gel was solidified for 1h
The gel was run at 60V for maybe 40min and with chilled 1x TAE buffer (made fresh form the 25x stock and it was still cool when the run was done)
The ladder is a 50bp ladder

Unfortunately it seems I can't upload the image

-Baconman-

Is there any difference in the way the samples were prepared compared to previous runs?

how does the ladder look?

if the ladder is also diffuse, then i would try with fresh buffer (not fresh dilution, there may be a problem with your stock)

check the pH of your diluted buffer

Do you always wait 1 hour before using the gel? Have you tried letting it set longer (even overnight)?

-mdfenko-

No, I am doing everything exactly the same. One of the samples is my positive control for the genotyping PCR (it's a heterozygote so gives two bands for both primer pairs) and has worked well many times.

The ladder looks ok, but I will try to make fresh buffer just in case. Would you use TAE or TBE?

I normally let the gel solidify for less than 1h. Have not tried overnight

-Baconman-

Now the site allowed me to upload the image.
As you can see the ladder looks alright, the lower bands are strong but somewhat blurry, the larger bands are weak and very blurry. I have previously gotten 4 clear bands at 249, 325, 701 and 780bp

-Baconman-

Try fresh loading buffer, make sure the load stays low (don’t let it get diluted by electrode buffer, load at the bottom of the well rather than the top) and start immediately after loading to reduce sideways diffusion.

after ensuring that the gel has completely solidified and cooled

-mdfenko-