You have 2 issues here but first the frowning. You could try loading less sample either as a lower volume or the same volume of sample diluted 1:2 with water ( this lacks density so will not load as easily) or make up some more loading buffer of bromophenol blue or orange G or xylene cyanol in 1x TAE with 50% glycerol or sucrose and use 1/10th volume of this for your samples and size standard. I do not think that the phusion buffer is directly causing this effect...it is widely used and tested. Are you losing volume in the pcr cycling which would increase the salts in the remaining solution. With 10ul reactions if the plate/strip is not spun down then there may be condensation at the top of the tube leaving the reaction mix as quite high slt so spin down before sampling. If another researcher uses home made or younger loading dye borrow some to test a size standard and/or sample.

The second issue is the smear that you report. It would be interesting to see a picture but a good pcr should not leave a smear in the gel. A smear suggests too much or degraded dna or that the pcr parameters are incorrect possibly with one primer not annealing well and the other producing random length smears of product, Possibly also the quality of your original dna is poor. If you run just a dna sample (not amplified) does it produce the smear in which case you are probably using too much dna  When you load your samples check that the sample stays in the well. Sometimes if a gel is poured too hot after melting the agarose the gel tray bends and the comb sits too close to the bottom of the gel tray. Then when you pull the comb out the base of the well tears and some of the sample can leak under the gel and then moves with the current and can look a bit smeary. Check also that other lab users are getting good electrophoresis results. It can happen that someone makes up the 1x TAE wrong from a concentrate or it gets contaminated and things grow in it and degradation of the pcr product can happen.

Lastly if you run too many cycles then the product can anneal to itself and produce dirty longer products. How much template and how many cycles and is your target a single copy gene?

Hi

I think the reason for that is the high voltage which cause that. It's better to decrease the voltage to 90 V or 60 V and see what happen then.

Good luck

Could be some contamination on the surface of your comb,  give your comb a good clean, first with detergent, followed by socking in 100% ethanol and then in water.

I think it is caused by too high salt in the sample compared to the gel and as the sample runs further through the gel the salt diffuses away from the sample and the sample eventually runs better as its local environment becomes more similar to the gel buffer composition

Thank You very much for Your advices and suggestions. I will make sure, the chamber and the comb are totally clean. I already decreased the voltage to 65 yesterday and ran for 95 minutes, but the smiley shape was still there.

Today I will check the salt concentration of my samples (buffers) and try again.

Again, thank You very much for Your help. I appreciate it.

agreed smear are often linked with contamination. In addition to the comb, make sure that you are using clean autoclaved water. That usually helps too.

Cheers!

So I performed all the steps again and ran the gel at only 60V for over an hour. Looks slightly better, but still unhappy smiley shape of the bands plus the smear (also a bit better). 

I was wondering, Paul Rutland, may I ask how I can address the possible issue with the salt concentration? I am using the phusion polymerase kit which needs to be used with a 5x phusion HF buffer. 2µl in a total 10µl reaction volume are used. For the gel I use 1xTAE buffer, also for running buffer. What can I change to avoid a difference in salt concentrations, as You suggested?

Thank You for Your help.

You have 2 issues here but first the frowning. You could try loading less sample either as a lower volume or the same volume of sample diluted 1:2 with water ( this lacks density so will not load as easily) or make up some more loading buffer of bromophenol blue or orange G or xylene cyanol in 1x TAE with 50% glycerol or sucrose and use 1/10th volume of this for your samples and size standard. I do not think that the phusion buffer is directly causing this effect...it is widely used and tested. Are you losing volume in the pcr cycling which would increase the salts in the remaining solution. With 10ul reactions if the plate/strip is not spun down then there may be condensation at the top of the tube leaving the reaction mix as quite high slt so spin down before sampling. If another researcher uses home made or younger loading dye borrow some to test a size standard and/or sample.

The second issue is the smear that you report. It would be interesting to see a picture but a good pcr should not leave a smear in the gel. A smear suggests too much or degraded dna or that the pcr parameters are incorrect possibly with one primer not annealing well and the other producing random length smears of product, Possibly also the quality of your original dna is poor. If you run just a dna sample (not amplified) does it produce the smear in which case you are probably using too much dna  When you load your samples check that the sample stays in the well. Sometimes if a gel is poured too hot after melting the agarose the gel tray bends and the comb sits too close to the bottom of the gel tray. Then when you pull the comb out the base of the well tears and some of the sample can leak under the gel and then moves with the current and can look a bit smeary. Check also that other lab users are getting good electrophoresis results. It can happen that someone makes up the 1x TAE wrong from a concentrate or it gets contaminated and things grow in it and degradation of the pcr product can happen.

Lastly if you run too many cycles then the product can anneal to itself and produce dirty longer products. How much template and how many cycles and is your target a single copy gene?

Thanks for Your detailed answer. I will make sure that all reagents and devices are totally clean. With other collegues the gels look nice. No smear or smiley shapes of the bands. But I will test some different settings of electrophoresis as well as chamber types, current and volt today as well as buffer preparations and dyes today.

I run 30 cycles of PCR with 1µl template in a total of 10µl reaction volume (cDNA was done from 500ng of RNA, so this would make a 1:10 dilution in the PCR of the template). The target is a variable chromosomal bacterial region. The PCR protocol was already established, that is why I do not want to change the PCR parameters. But maybe it would be best to do a gradient PCR to check for some better annealing temperature.

Before PCR reaction I always spin down the tubes to make sure nothing is left on the tube walls or the lid. And I am sure I am not losing volume during PCR, so the salt concentration should be fine. 

Another problem I have is that my RT- control of the Reverse Transcription is always slightly positive, but the visible band runs always a bit higher than the  band of interest (around 30-50bp). Are these genomic DNA contaminations in the cDNA samples? The negative controls and PCR water controls are negative.

If other workers samples run well then  borrow some of their loading buffer to compare to yours. The pcr sounds very good but a gradient might clean it up if your pcr machine calibration is different from theirs but that is just a detail.. I am sure over amplification will not be an issue and clearly neither is pcr contamination and I think you are probably correct about genomic dna amplification. Good luck, Paul

in addition to all the above suggestions, Just make sure that since you are running the gels in 1xTAE, make sure that while preparing the agarose gels, you are using the same final concentration. In our experience, we face lesser problems with TBE buffer.

best wishes,

SD

Thank You very much for all Your suggestions and thoughts. I really appreciate them! I will get back to You when I have found a solution :)

I usually start all my gels at 50 V and wait at least 30minutes before increasing voltage. Also, try running the gel in the cold room. 

Hi Anna,

If your ladder is fine, so your DNA amount is high.

-If the ladder is also smiley than I recommend you to change your running buffer from TAE to TBE. TBE has better resolution for short runs with high voltages (1-2 hours at 80-90 volts). TAE causes high heat at high voltages and this can ruin your bands.

-Use always homogenous or fresh loading dye buffer, it means you have to vortex it before use. Otherwise, the salts in the buffer precipitate during the long RT storage. 

-Always prepare 1xTBE fresh due to salt precipitation again.

-another suggestion, you should balance your agarose gel (remove the comb from the gel) with your running buffer at least 20-30 minutes before loading your samples.

-Before you load the samples, clean your wells with running buffer by using Pasteur pippet to avoid any remainings.

-By the way, when you prepare the agarose gel, you have to make sure your gel homogenous.

And when you do your PCR, you have to make sure your enzyme completes all the fragments. To do so, you have to use longer extension step (if you use 30 second, for last circle use 90-120 sec) at the final circle.

Good luck!

If your PCR product is more than 500bp so i will suggest going for 1% gel instead of 1.5% as this may cause the bands move easily in the gel when pushed by the negative charge. The less saturated the gel the more it will allow the larger fragments to pass easily 

Try loading the loading buffer in empty wells and also check for glacial acetic acid in TAE buffer used.

there are several factors may affect the results, if the ladder and PCR product produced smears, so you need to change buffer to TBE and checking its pH and the temperature, also you can decrease the concen of agarose if your product more than 1000 bp, third you can use a 100  starting voltage for 5 min then deceased to 75-80 for one hour. finally, if you show me the pic can give you more accurate suggestions.

Smiling and frowning is differential electrophoretic mobility across the well. This is usually caused by differences in solute conditions. High salt concentrations (and, remember, DNA itself is a salt) may increase or decrease the mobility. At the edge of the track, the salt can diffuse out of the sample, and, in your case, permit a faster mobility. In the middle, the salt conditions stay higher for longer. 

It is quite possible that the smearing problems are due to salty sample as well. 

Running some sort of cleanup ought to help. One of the post-PCR cleanup columns ought to work. Ethanol precipitation followed by washing the pellet in 70% ethanol may help - but make sure you get rid of all the ethanol afterwards. 

One trick that might work is to start the gel slowly (15-30V) and run it just long enough to get the sample into the well, then turn off the current for an hour. This should give the sample time to equilibrate with the gel buffer. I've never tried this, but in theory it should work. 

Thank You for Your suggestions!Our product is approximately 450bp , so 1.5% gel should be ideal concentration. We use sybr green staining.

We were not able to fix the shape- Problems and the smeary samples and markers although we tried all different buffers, voltages, cleaning of Chambers and combs as well as PCR dilutions. But now we used a different chamber with the same buffers, voltages, times and load, but just a different device as well as HD Green Plus Gel stain instead of SYBR Safe gel stain. And now everything Looks perfect.

The Smileys are gone as well as the smear in the marker. The smear of the RNA and DNA bands disappear when the PCR products are diluted. The bands are perfectly straight. Ist also the same 1x TAE buffer receipe as well as agarose and gel percentage. Just different device and gel stain in a different Building and voila: it worked. We still dont know why it didnt work out before.