Noha,

There are two possibilities you should consider: 

1- The DNA concentration after the extraction was so low that you cannot see the signal after staining with the intercalating agent, but the DNA is there. So, after PCR you amplified a fragment from the DNA , and generated a higher concentration of amplicon products than the original DNA, then you can see the bands. 

2- You do not have DNA at all, and you ran the PCR reaction. Maybe , without a template your primers can originate primer-dimer products, so you will see amplified small fragments, but they are nonspecific. 

To solve these questions is important that you have positive and negative controls in your PCR reactions. 

Andre

Noha,

There are two possibilities you should consider: 

1- The DNA concentration after the extraction was so low that you cannot see the signal after staining with the intercalating agent, but the DNA is there. So, after PCR you amplified a fragment from the DNA , and generated a higher concentration of amplicon products than the original DNA, then you can see the bands. 

2- You do not have DNA at all, and you ran the PCR reaction. Maybe , without a template your primers can originate primer-dimer products, so you will see amplified small fragments, but they are nonspecific. 

To solve these questions is important that you have positive and negative controls in your PCR reactions. 

Andre

HOW CAN I HAVE POSITIVE AND NEGATIVE CONTROL IN PCR?????

Hi,

There are many tricky points in DNA extraction from blood, first: it is preferred to work in fresh blood not freezed blood if you do not use good quality extractions kits like qiagen kit. second point you may be concentrate blood sample by working in buffy coat  in this case you will have a lot of DNA. third point: it is preferred to work with column based extraction kit to have consistent results. when you make electrophoresis for extracted DNA you will find smear not bands, this smear indicate good DNA yield.

Where the bands correct in size or melting point? Primer dimmers are almost ubiquitous when PCR is pushed to hard, to high primer concentration, and it could be your original sample as a contaminate.  Never under estimate the power of PCR. 

Another possibility was that your PCR was not optimized for ultra low detection and just needed more cycles.  Many weird one time results occur with PCR, my advice from the using PCR before the advent of thermo-cyclers is do not dwell on artifact unless can be repeated.   You have better things to spend your time on.

Hi Noha,

1. Without visualizing the DNA on a gel doesn't mean there is no DNA there. Probably your DNA amount was too low to be seen on the gel. But, PCR is a very sensitive and powerful DNA-amplifying tool, so you can see bands (amplicons) of PCR product on gel later on. 

Please see the discussion of the same topic on RG here: "What is the minimum DNA quantity (ng) required to visualize DNA on Agarose gel"? 

https://www.researchgate.net/post/What_is_the_minimum_DNA_quantity_ng_required_to_visualize_DNA_on_Agarose_gel

2. Make sure the PCR band (in all your samples) amplified is your intended target, not just a non-specific amplification product. If this band also appeared in your 'negative control' (PCR reaction without DNA template [replaced with water]), I will be worried for some kind of contaminations.

Hi again Noha,

It seems like we face same issues except I work with environmental samples and you are not. Anyway, if you want to know if your extraction has any DNA or not, measure them with nanodrop. They can give you what the concentration and you can look at the ratios of 260/230 and 260/280 for the detection of possible contamination. The measurements are not that accurate with

I would agree with others that your native DNA concentration might be too low to show up on a gel, but there is enough DNA to PCR.

You don't mention the size of the bands you get on PCR, but I would expect primer diamers to run very quickly on the gels, usually very close to the dye front.

I think Nanodrop would measure even very short fragements - however it is a quick way to see if you have something there.  There are other DNA quantification methods such as picogreen, qubit and sybrgreen. Some of these use dyes that lock into the DNA helix, so need a reasonable length DNA fragment to work.

I think the DNA concentration of the extracts was so low, so didn't appear on the gel. But after PCR the amplification was succeded so the DNA bands appeared on the gel, as the PCR reaction is very sensitive for very low DNA amount.

Wishing this is helpful.

Best Regards

Negative controls: Water instead of template should not give any signal except for primers and primer-dimers. If your get more than that, you have a contamination in one of the reagents. What you can do additionally: leave out the primers separately to see if the primers do weird things. You should get your template band and primer bands. If you don't see anything in your controls, it's probably a matter of concentration, don't worry about that.

Positive control: known set of template+primers working at the conditions chosen for your PCR. This control is for the enzyme and for the PCR run, if you don't see a correct band, there's a general problem in the PCR, not only with your DNA. This works especially if you use the same primers and conditions for several templates, because every primer-template set will have its own optimal conditions.

Hi Noha,

Like the others answered, it's most likely that this happened because DNA concentrations were low post extraction and much higher post-PCR. I think the best thing to do, if you need to know what concentrations of DNA you're getting, is to quantify your DNA using a nanodrop.

Good luck!

You will not obtain any band except if your reaction is contaminated  by some DNA similat to that you want to amplify

I'm agree with dr Abitbol, you will obtain an amplified product only if there is contamination by some DNA similat to your template. Anyway, you can perform a negative control in your reactions.

You need always a negative control. As Andrè Luis da Costa-da-Silva says you can see oligonucleotides dimer if there is no amplification of starting DNA. 

You may have some contaminations in your reagents and plasticwares .This problem may also be resulted from nonspecific primers which can anneal to and amplify very low amount of DNA in your samples .

I agree with above discussions. it might be primer dimer or DNA contamination. add negative control and see results, also change your PCR reagents use fresh one aliquots and take proper precautions while preparing master mix and adding template to avoid contamination.

i concur with the 3 possibilities, low concentration of DNA before PCR, and possibility that the bands after PCR are either primer dimers or contamination but most likely dimers since contamination may not be uniform in all the samples. Just include a negative control to establish this.

If you don't add DNA, you may see primer dimer and if reaction is contaminated with DNA it will give band in all tubes including blank.

I think you have tow reason

First   your DNA quality not good. Second   your primer not good so you should try again carefully.

Good luck