The possible cause may be due to DNA diffusion in the gel,avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion of smaller DNA fragments in the gel.

Avoid long term storage of the gel before taking a picture, as this may cause diffusion of DNA fragments and low band intensity.

Dr. Jameela 

Thanks for your suggestions, but I use the same stain every time without any problem, except at that time. In addition, I expect to get one band with the same molecular weight as the one in lane 2 (around 8.5 kbp). Can this molecular weight diffuse to the gel with extensive electrophoresis time?

Dr. Liu 

Thanks for reply. The only difference between bands 4 to 8 is only in the incubation period with the releasing acidic medium.

I will check if there any protein contaminations in the samples or due to the comb although I clean it with ethanol before using.

I added the dye to agarose solution before casting.

Dr. Roy

Thanks for reply. I took this possibility into consideration. However, the software still can detect bands on the expected positions and the amount of liberated DNA follows an expected sequence.

Dear Amir,

In addition to all points raised by all researchers, i want to stress on the starting concentration of DNA linked to nanoparticles. I recognized that the band 2 gives altougth well defined band of original DNA but it is not that of high concentration . SO, we expect that the released DNA from the nanoparticles is at so low concentration to be undetectable in the applied volume to each well as it might be diluted or released at low concentration.you need to increase the volume or concentrate the sample before application to each well or to increase the original DNA sample linked to nanoparticles.Also with different incubation temperature you have to ensure protection to your DNA from DNases by adding EDTA to the eluting buffer and be aware of temperature of the eluting solution as it also affects the release of DNA from these particles as there is more than faint two bands in well 8.Are you using ethidium bromide or other stain? as i noticed some dark shadows before and after the shadow of DNA which we find them somtimes when we use ehtidium bromide and this needs to be reduced by adding some ethidium bromide to the running buffer to minimize this .Consider the use of new freshly prepared running buffer and reducing the time of the run.  Good luck

Dr. Najat

Great thanks for your suggestions.

Today, I have tried higher concentrations of the DNA (from 125 to 200 ng), but also didn't get any clear bands. In addition,  higher loading amounts of the DNA to the nanoparticles couldn't make intercalates at the the experiment design.

I will try to add EDTA to the elution buffer for inhibition of the DNAses.

Moreover, a major problem with this gel as you mentioned is the dark shadow due to ethidium bromide. I haven't observed it before, but all my current gels have it, so I will follow your suggestion regarding it with using fresh buffer.

also i want to know the % of agarose and the details of electrophoresis conditions and type and PH of buffer. Did you neutralize the sample PH prior to electrophoresis as you used acidic medium for incubation?!

I use 0.5 % agarose. Previously, I used to use 1 % agarose, but for faster running, 0.5 % is used. The voltage is 80 and running time between 40 and 60 min. Max. The buffer is TAE and its pH is 8.2.

I neutralise the samples using NaOH and test the pH before sample loading using litmus paper as the sample volume is small. For comparison, I run samples using different volumes of added NaOH, but also got no bands.

Are you running the solution into which your DNA was released? If the DNA is conjugated to your nanoparticles, I don't think it will run. If this is the release solution, perhaps there is significant dilution of your DNA in the solution? I do agree with above that I see a DNA band in the last lane on the right. It could be a matter of concentration. How much DNA did you use in well #2?

Dr. Hongye

The release study was performed before on the DNA/nanoparticles conjugate, but on different gels and I got bands with different intensities from 4 hours incubation forward. I use the release solution directly for running gel as it will contain the released DNA, and got previous results. Yes,there's a band in the last lane, but it's weak and the other lanes don't show any bands as before. 

In the second lane, 0.1 microgram DNA was used corresponding to the expected maximum released DNA.

how acidic is your release medium and how long is it after elution that you neutralise the eluate.Like Najat  I am concerned that too long storage in acid medium before  neutralisation may be depurinating your dna

Dr. Singh

I only used agarose gel electrophoresis. I have obtained clear bands with different intensities before at the same different times. Sometimes I get also the fluorescence in the wells due to the residual DNA in case of the conjugated DNA without any releasing medium.

please, how can I check the polarity in that case? 

Dr. Rutland

I use HCl (0.01 M, pH 5) for releasing of DNA as the nanoparticles are composed of layer double hydroxide. I even used HCl (1.5 M, pH 1) for few minutes to test the release of DNA, but obtained faint bands unlike expected.

For neutralization, I tested different volumes of NaOH for different times (1-5 min), but couldn't get differences in bands without detecting any bands as well. 

Regarding the period of incubation in acidic medium, the 8th lane (24 hours incubation) shows partitioned fragments at the Mol. Wt of interest; however, for the shorter times, no bands were detected. That really makes me crazy, and the software can detect bands with the same Mol. Wt. at the different times although they can't be visually observed.

dear Amir, you may want to check your arithmetic on this one. The pH of 0.01M hcl will be pH 2  which is very acidic and may effect both depurination and neutralisation.Does this make a difference?

Dr. Rutland 

I just diluted stock HCL  (9.6 M) with water then adjusted the pH to 5 with few micolitres of NaOH. Before loading the samples for electrophoresis, I neutralise with NaOH and test the pH till becoming 7 for running gel. 

In general, this is my second trial with the same HCl. In the first time, I got bands whose intensities were as expected within 4 differeng runs.  Now (2nd trial for the concluding the whole my previous work), I can't observe any thing.

a possibility is that NaOH absorbs CO2 from the air to form Na2CO3 which will have a major effect on its ability to adjust pH unless it is made up fresh each time and might account for it working once but not the nest time if the storage allowed air into the NaOH between experiments.

Dr. Rutland 

Thank you for referring to this point. In the next time, I will prepare fresh NaOH in decarboxylated water, and test its efficiency. That may be the reason for the absence of bands under the acidic conditions.

Another issue is regarding to one of the bands resulting from the centrifugation  of the nanoparticles/DNA conjugate. Previously, I used to centrifuge prepared conjugates for releasing all their contents of the DNA which used to appear as intense bands containing similar amounts to the standard DNA, but now no bands can be detected at all even with using different centrifugation speeds/periods. I just take the supernatant and run directly in gel. Please, could you help me with issue?

Hello Amir,

I am not familiar with this technology but if the unreleased DNA remains on the particle during electrophoresis perhaps you could arrange the experiment so that you could load a non separated part of the whole reaction mix (adjusted to about pH8) with the current leaving the particles in the well and running the DNA through the gel. As you are not getting release of the DNA under similar centrifugation conditions generally I would check the DNA concentration before and after binding to make sure that some DNA is binding to the nanoparticle and then again after release using a suitably zeroed spectrometer/nanodrop although degraded DNA will absorb also after release under these conditions.

As it has worked before the only thing I can suggest is to get your acid eluted DNA neutralised to alkaline ( about pH8) as soon as possible after release from the particles  possibly using a solution with a little EDTA in it to also ensure that there is no nuclease activity  in your dna solution while you store it prior to loading on to the gel

Thanks Dr. Rutland for your help and advice.

I will try to measure the DNA concentration using spectrophotometer in all cases, and modify my conventional procedures using EDTA.

Dr. Artur

Thanks for the explanation. I have tried adding a small amount of theEth-Br to the buffer, but without improvement of the image. Now, I will use the same amount in the gel and buffer.

Regarding the loading dyes, it contains 2 dyes, one of them has a Mol. Wt. of 4000 and the other one has very low  Mol. Wt. Although the studied plasmid results in a band with Mol. Wt. of 10.5 Kbp, I will try with another loading dye. 

Dr. Ali

Based on the explanations described before through all replies, I have started now to adjust the pH to 8 exactly using freshly prepared NaOH to neutralise the HCl in the releasing medium with monitoring the effect of the incubation period in HCl on the integrity of DNA and bands.

Hello Amir,

The greatest breakthroughs in research come from trouble shooting something like this. Do the nanoparticles prevent the ethidium bromide from intercalating with the DNA? Quick and easy way to test: add some nanoparticles (or even better, some sample)  to your DNA MW ladder and run gel as usual. Compared to untreated ladder, if you can not see the treated ladder anymore, you have a clue to the problem.  Then make incremental variations until you get EtBr staining of the ladder again. Alternate control would be to use some DNA that you have in abundance and try variations of pH, concentration of salt (due to adding NaOH). Try to mimic the experimental conditions knowing how much DNA you have, and learn when the staining is inhibited.

I look forward to your discoveries!

When I study your gel, it looks like lane 8 has a very faint band of the size of your lane 2 DNA. Is this the longest time point? Perhaps it just takes longer to elute the DNA from the nanoparticles?

Dr. Cynthia 

Great thanks for your reply. 

The nanoparticles make intercalates with the DNA based on the ionic binding between the phosphate groups in DNA and the positively charged surface groups in the nanoparticles, so the macromolecules won't be free to bind with the Eth-Br, so can't be detected till becoming free.

Regarding the testing of different conditions of pH, concentration and salt, I have tried them but with the DNA/nanoparticles and changing the concentration as well as the incubation periods with EDTA, HCl and NaOH based on the explanations presented in the previous replies, and now continuing these studies. Please, what will these results refer to (with DNA only)?

For the 8th lane, yes it's the longest period of incubation with HCl I have tested till now.

Dr. Ali

I have summarised how the DNA binds with the nanoparticles in the last comment. I also have just tried this intercalation in the presence of EDTA based on the previous suggestions as the presence of DNAses may be responsible for the degradation of the DNA, and some bands started to appear after incubation for 4 hours. However, the all procedures still require more optimisation.

Dear Amir,

To mimic the experimental conditions I should have been more clear. The nanoparticles would need to be included also.

Dear Amir,

If as you said that there is some improvement after using EDTA, so it means that there is ongoing DNA degradation. So, i suggest that you have to prepare all used solution in DEPEC water.DEPED stands for diethyl pyrocarbonate and it acts as nuclease inhibitor and in molecular biology experiments we use a term called nuclease free water and is available commercially.This water can be prepared as 0.02%-0.05% in DW and left at room temperature for `2hours and do autoclaving for 15-20 min to remove the traces of DEPEC and the resulting water is used in the preparation of all needed solutions except for Tris containing buffer or reagents.It is important to you to use this water even in washing your nanoparticles.I hope this will give you better results in addition to EDTA. Hopefully to send me the gel photos later on to observe the changes. Good luck

Thank you, Dr. Najat

I have started to use this water in the new trials as well, but I use the commercially available water from Thermo Co. without any further treatment as you illustrated.

For the washing of nanoparticles, I use decarbonated Millpore water. As large amounts are used, I haven't tried the nucleate-free water; however, I will start to use in preparing the suspension of nanoparticles for the intercalation steps.

Once, I finish these release studies, I will upload the new gel images.

Thanks for all your suggestions.

I have tried different concentrations of EDTA during the intercalation of DNA to the nanoparticles and found the optimal one which was used further, and adjusted the pH after the release of DNA in HCl medium to be 8 using NaOH (adjusted concentration and neutralization period), then Eth-Br was added to both the agarose gel and the buffer (with pH 8), and finally got the attached picture. However, the lanes corresponding to the release (4-8) showed lower molecular weight for the released DNA. Something may have happened due to the added molecules (EDTA, HCl, NaOH) which lead to these irregular bands.

The major fraction according to the supplier is supercoiled, and a very small fraction as linear. The linear one sometimes appears in addition to the supercoiled naked DNA, but here didn't appear (lane 2 and 3). 

Dear amir

Although i still do not know your aim of the study, but definitely there is good improvement in your DNA gel yield . the presence of two bands may reflects the possibility of DNA  melting or you may called it relaxation.the presence of high salts concentration make a shield to negatively charged phosphate groups in the DNA and affects as well their mobility,so, if you adjust the salt concentrations in the samples presented in lane 4-8 to be similar to those in lane 2-3 probably you can get similar pattern. May be you need to do one of the following:

first if you have a minicolumn kit for DNA extraction so you can use the column and apply your sample to it and do centrifugation to get rid of salts and the DNA will be adsorbed to the column resin . Then use the washing solution of the kit to do 3-5 washes of the column to get rid of extra salts and then use the 25-50 microliters of the eluting solution of the kit after drying the column by centrifugation and use the eluted DNA solution for a new electrophoretic separation and observe the effect.

Or you can apply your DNA sample on a small column packed with sepadex G-25 to desalt it and them apply it on gel. I hope these suggestions will help you.

. Good luck

I will try using the DNA extraction kit, but the sample volume after all treatments is initially 10 microlitre.

Yes, I tested the effect of Kpn I restriction enzyme on the DNA and intercalates and got the bands on the expected positions as you explained,  Dr. Ali, but in the case of HCl treatment, the molecular weights were lower than the expected weights.