DNA PAGE - Why are my Bands and Ladder smeared/Not Clear?

I did PCR of 6 amplicons and when I did agarose gel electrophoresis, they all appear consistently bigger than they should be (e.g. the amplicon should be 4000 bp, but is around band of 5000 bp marker).

Sizes of my amplicons, from left to right are:

top row - 2610, 4052, 4000

bottom row - 4860, 4565, 3235

The pictures are the same, just different exposition. There are 5 identical samples differing at annealing temperature, split by marker.

The marker looks quite bad. I don't know why, I used it previously. The conditions of electrophoresis are: 1% agarose with TAE; 50 V; 90 mins

I want to do PCR amplification with my full-length gene and the addition of P2A fragment at the end of my gene. However after doing PCR, I run gel electrophoresis for analysis. But it doesn't include the band for my gene. I run the same template DNA with other primers for smaller fragment, and it has the band. I tried to redesign my primers for full-length fragment, but it still don't have the band for my gene. Can anyone help me? Thank you

Hi, I ahve tried dna extraction and when I load DNA in agarose, I have seen dimer like dimerprimer in PCR, can anyone tell me why I saw dimer in dna extraced?

What are the 3’ end modifications that prevent DNA from being extended by DNA polymerase? Which one has the best blocking effect? Leakage is minimal?

My fellow Academic colleagues!

I together with my lab mates have a PCR-related issues that we hope that some(one) of you might have encountered and hopefully solved.

In “short”, our initial PCR (MiniAmp Plus thermocycler) and electrophoresis protocol works like a charm – the latter somewhat modified. We obtain weak to strong band that yielding concentrations of 9 to 20 ng/µl following clean-up using the QIAGEN QIAquick PCR (& Gel) purification/Cleanup Kit (with an acceptable A260/A280 ratio). We obtain rarely, but from time to time, a positive electrophoresis confirmation. But as we are using the same protocol for the confirmation, as for our initial PCR, we should have no issue confirming our results (one band per week).

Usually, when we try to confirm our cut-out electrophoresis bands, running a PCR on our cDNA, something fails. We utilize the same primers and protocol, as for the initial PCR, but nothing shows up in our gel, our at best a streak. We’ve tried renewing our primer mix(s), new isopropanol, new buffers, using both RNAse-free water and the included buffer, modifying temperatures (thermocycler), number of cycles, and using the original non-modified protocol. But nothing results in an electrophoresis band when we try to confirm our initial band.

Thank you for your insights and help!

// Eriksson et al.

I ran PCR using COI universal primers on DNA extracted from lice.

I added 25ul of 2x master mix, 5ul template, and 1ul each of 20uM F and R primers, with the remaining volume made up with DW to a total volume of 48ul, and ran PCR including a control group. However, no bands appeared on the gel after electrophoresis.

I then checked with a nanodrop, and all 5 PCR samples (including the control group) showed concentrations around 20000ng/ul, with A260 readings around 400, 260/230 ratios around 10-11, and 260/280 ratios around 37-47.

Where could I have gone wrong?

I would appreciate input from experienced individuals.

I have a recipe of loading dye (10 mM EDTA, 1X TBE, pinch of Xylene cynol and Orange G dye which is made upto 10 ml with 100% Formamide) but somehow the bands of the nucleotide product (enzymatically degraded DNA) are not distinct and well defined or fuzzy. I use 1 X TBE buffer (freshly prepared), the volt used to run the gel is 1500-2000 Volt or 25-40 W. Could you please suggest the recipe of loading dye or other factors that could give crisp bands for publication purpose. Any advice would be highly appreciated.

Thank you in advance

Prem

Maybe someone knows why this happens. The situation is that after PCR purification of gel products (cut one band), on the next electrophoresis, instead of one band, two bands appeared, how could this happen?