The gel looks fine. It just looks to me like 3 successful PCRs with a lot of starting template, three successful PCRs with less starting template (or reaction inhibition), and then six unsuccessful PCRs. Is it possible that samples 7-13 simply don't have any template? Or are sufficiently packed with inhibitors of PCR (guanidium, humic acid, etc) such that you don't get any amplification?

May be increase the concentration of agarose gel 0.8 to 1%. Also, You need to check the concentration of samples

agarose gel 1.5 %, 100V in 30 min.

If your ladder is showing up on both sides, you can be reasonably sure it's nothing to do with your gel or staining (an easy test would be to simply load the same samples in reverse order), plus gel electrophoresis is relatively difficult to really screw up (any agarose conc between 0.5 and 2% should be more or less fine, and voltage just needs to be high enough to get DNA to move, while not so high that it makes your gel melt).

So: my suspicions would then point at your PCR.

What are you PCRing, what are your different samples (different samples, different cycle conditions, different concentrations?) etc.

Also, pictures might help.

Hi,

there may be problem in electrode wires. there is wire on each side in the tank which generate electric potential difference. You see if small bubbles are there at one side (cathode ) in the tank along with the submerged electrode wire. if it is unusual then it need to be repaired.

good luck

Thanks dear researchers....I am trying to amplify 16srDNA of LAB isolates. I am using similar three steps PCR conditions for all the samples. Also bubbles are being generated at the cathode all throughout. I am attaching herewith a photograph for better understanding.

The gel looks fine. It just looks to me like 3 successful PCRs with a lot of starting template, three successful PCRs with less starting template (or reaction inhibition), and then six unsuccessful PCRs. Is it possible that samples 7-13 simply don't have any template? Or are sufficiently packed with inhibitors of PCR (guanidium, humic acid, etc) such that you don't get any amplification?

the gel looks ok, probably DNA loading well is not retaining the DNA, because at times due to rough surface or imbalanced comb the wells show leakage at bottom this can be observed while loading the sample with dye.

secondly most probably its PCR failure....

Nothing wrong with the gel or gel running to my opinion. As someone else pointed it is the result of an unsuccessful PCR in your lanes 7-13. Try to modify the PCR conditions but check first if you have or how much DNA (including quality) you have isolated and used as template from lanes 7-13 compared to e.g. lane1. Use a positive control lane 1 or 2 in your next trial for samples in lanes 7-13.

in my opinion the problem might be thermalcycler - problem with equal temperature in the whole block. Try to use deferent PCRmachine

If  your DNA ladder is ok,  you may take the positive and negative electrodes in wrong way, or  agarose gel  is Failure.

Hi, two questions: how did you stain your PCR products? by adding Etbr to gel before polymerization or immersed your gel in Etbr solution after gel running, Did you try to put the 7-13 samples in the 1-6 lines? 

Hi Arup,

Seeing the gel picture I guess there is no problem with your work. PCR and wells in the gel are looking nice, staining is ok. Please try loading samples in gel in opposite direction (start sample 13 in first well).

There might PCR with missing DNA. Try another PCR carefully for those samples.

If sample 7-13 still do not give bands there might be the reason that those samples do not have target gene. Once of my colleagues faced the same problem and he repeated the process for more times and did not find bands. Finally he concluded them as separate group.

Hi Arup,

Gel looks fine. So my suggestion is to do PCR again for those samples showing no bands in gel.There might be failure of PCR. Try, if needed, different conc. of primers, templates, different annealing temperatures etc.

Thanks,  

It looks no  problem with the gel and staining. but as some others pointed out the gel might be leaking at that area or 7-13 lanes lack the template. so try again with some changes in PCR steps.

Actually, nothing is wrong. Your DNA might be getting amplified also. Just check the loading of the gel. Do not alter any of your parameters like agarose concentration or the power supply. However, a remote possibility of the other DNAs is that there could be shearing during purification or in some other processes. Just try to run a simple agarose gel electrophoresis with those DNA which are failing to  show up during your PCR study.

Hi Arup!

In my opinion and experience, there is nothing wrong with your gel or gel running. I think that there might be missing DNA in those samples. First check how much DNA and  its quality that you have isolated. Repeat PCR  for those sample and use a positive control. If these samples continue the same way by not showing DNA bands in gel, the reason might be that there is no target gene.Good luck!

Before you separate your PCR amplified DNA did you measure the concentration of the DNA by means of Nanodrop (260/280 nm, 260/230 nm) ? It's very important to estimate the concentration of the DNA in order to optimize the concentration of the agarose gel and perhapes you didn't get bands because of the lack of sample

Hi Arup!

As previouly mentioned, technique seems to be fine. I would change the loading order, and check if it is a problem from specific samples (it seems so). Then we can figure out what happens with those samples ;)

Let us know how it went!

I would highly suspect an issue with the thermal cycler, especially if all of the templates that you used are similar (not different concentrations, etc.).  The pattern looks like there might be a thermal gradient across the cycler--or one side of the block might be "dead".   I would try a different instrument or, if none is available, try your reactions again in that part of the PCR block that gave you good product yields.  Your gel looks fine.