possible explanations are.

1)The concentration of DNA(PCR product) loaded might be very less.

2)it must have run out, check the size of the products

3)Did you add sample loading dye(which gives density to hold the DNA in the well)

If the RT-PCR is working, the DNA is there but it is probably at very low concentration - too low to see the fluorescence on a gel. Often PCR actually works better at lower DNA concentrations. You should be able to quantify it with fluorometry, or with a UV-spectrophotometer (although that is slightly less accurate and usually requires a larger sample).

Or it's possible that you are amplifying something other than the target sample in RT-PCR. You might want to make sure you don't have a contaminant somewhere.

possible explanations are.

1)The concentration of DNA(PCR product) loaded might be very less.

2)it must have run out, check the size of the products

3)Did you add sample loading dye(which gives density to hold the DNA in the well)

Assuming that you did not mistakes in loading the gel (this happens), I would say that you amplified only very little DNA. All optical PCR systems have a much higher sensitivity than agarose gel based detection systems. Are your samples coming up late (=have a relatively high Ct-value)?

First you have to check your level of contamination, Have to check your the quantity of amplified DNA using nano-specrtophotometer as the specific quantity of DNA is required to have the better resolution on Agarose gel, Also make sure that whether your target DNA is amplified or not as other target may also amplified that may show cross results.

You are not getting the amplicon, optimize the annealing temperature, for proper annealing of primers.

you can check DNA quantity by nano drop after amplified product.

Did you perform melting curve analysis after running real time PCR?

If yes, did you observe a specific product?

Try different annealing temperature, Also, decrease the percent of Gel that you used. Also you might decrease the time of running as your DAN might float and drop down in the buffer

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Please use a 2.5% agarose gel and increase the amount of DNA that you load into the well.

taken together the above comments, I agreed with them, in the beginning, ensure that your samples have a DNA material, then when loading the amplified PCR product into wells, attention to increase the volume in comparison to the size of well of the gel as might be you loaded little non enough to produce detectable bands or there was a problem in PCR protocol

I would like to agree with above comments. My advice is,

1) Please check your yield product DNA contents

2) If possible run the PCR again and check DNA quantity

3) Don't let your gel become too hard

4) Don't forget the loading dye and put higher concentration of DNA

All the best.

Hi

in addition of confirm that all of answers are exact, I think maybe because of your DNA are fragmented into the very small fragments.

Very rightly you did not get any DNA bands on carrying out the routine PCR amplification method. The simple answer is that it failed to amplify. Your q PCR technique seems to be ok and there you are getting the bands.

u need to

1- be sure u follow the right steps of extraction

2-u need to eliminate the ethanol from your samples

3- maybe your polymerase do snot work

5-programmed your PCR

4-sould made Nanodrop maybe many junk in your sample ,check purification

5- be accurate in loading amount of your pcr product on gell

6- many reason you need to check each step but Nanodrop will tell u ther's DNA or not ,is it pure or not if you have a good amount of DNA acording to your protocol but non pue you need to purification your sample by dialysis or other ways , if nanodrop tell u your sample is OK then the problem in PCR preparing or Loading way

all comments are right and I agree with them

You said your RT-PCR worked fine. But when you ran PCR that your DNA did not show up on agarose gel.

Does that mean that when you used RNA as a template the reaction worked, but when you used DNA as a template the reaction did not work?

If so your primers may span an intron and will not work with DNA as a template.

See http://www.protocol-online.org/biology-forums/posts/6379.html

The DNA can be visualized using a 3.5% agarose gel and use a suitable marker which starts from 25 bp onwards. Most of the RT products are within 200bp.

HI Abdul. PCR work is at times tricky, challenging ad at times stressfull. I agree with all the above comments. Your rt PCR worked, then most probably your samples have genetic material. I would suggest that you try and make slight adjustments in your DNA PCR reaction. When it comes in the stage of visualizing the amplified genetic material, check the reconstitution of the loading dye and try running several gels with different percentages, though this is just a contingency measure since your ladders/ markers are working well.

Good Luck!

Thank you. Your protocol may be right but you should check/quantify the conc. of DNA amplicon. As PCR is very sensitive but you may not get proper bands on gel if you dont properly use proper quantity of Target DNA. On the other hand, Better to check the loading dye used in the well whether is properly added with amplicons or not. Also depends on gel percentage..

did you determine the concentration of amplified DNA to be sure that it can be identified on agaros gel?

I think more information from your side will allow us to help you much better. As mentioned already there are a lot of factors which may be the problem.

A good thing to start with is what is the CT value of your product? If it is very high (over 35 I'm guessing?) it may not show properly on agarose gel.

A silly thing but you could have forgotten: loading dye for your sample.

A third option I'm thinking of is: you cannot be sure what you're amplyfing, could be primer-dimer formation (which may run off the gel if you run it too long). What is the expected size of your amplicon?

Does the meltingcurve of the PCR correspond to what you expect for your amplicon? (you can estimate it at e.g. https://www.dna.utah.edu/umelt/um.php)

Is your loading dye hand made, or from some manufacturer? You can ask colleagues, do they have similar problems, or not, and make the new dye, if it is needed.

How much DNA do you use for electrophoresis? Human eyes and gel doc are substantially less sensitive than rt-PCR.

hi,

your problems may be the following reasons,

1. Your extracted DNA sample having low concentration that may not be getting amplified in the PCR reaction. So, increase the template DNA concentration.

2. Please check your PCR conditions standardized or not . Mean time check your Positive control is amplified with your target product length without unspecific band.

3. After PCR amplification, you make a gel based on your final product length how much base pair accordingly you have to prepare your gel like the conc. 1%, 2% and 1.5%.

4. Use the EtBr conc. 0.1 to 0.5 μg/ml. Mean time Gel loading dye conc. also important. If it is high or low means when you loading the PCR product onto the well it can spill out then no more PCR products are in the well.

5. DNA was electrophoresed off the gel - Electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel.

6. Make some changes in the PCR conditions mainly primer annealing and primer extension step. Please make sure all the PCR master mix chemicals

are suitable concentration of your targeted bacterial genome and primer conc. is more important.

http://www.imbb.forth.gr/groups/minotech-new/pdf/troubleshooting_guide_for_the_electrophoresis_of_DNA_markers.pdf

Please go through the troubleshooting guide

Hello, Check the loading dye and try to run several gels with different percentages of agrose gel conc. and then visualize the band.

Replace the TE with DD.water and use a dye for loading the DNA

I agree with Christian Praetorius

Here you can see some guide

https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-troubleshooting.html

Check also this wonderful pdf