Hi

first of all, running of your gel face up with some problems because your ladder marker seems smear too.

dual are all your samples same or different?

do you check your samples DNA concentration?

good luck

I do not have experience with gelred dye. Nevertheless, there may be a background lighting as you ladder is also slightly lighted up. However, I also noted that your PCR conditions use way too much of components especially MgCl2 and dNTP and primers required for amplification using Taq DNA polymerase. The amount of components you used is a general reaction for proofreading polymerases optimized for amplification of high GC and these reactions usually involve a denaturants like DMSO in the reaction buffer. in the absence of DMSO, the high Mg++ and dNTP causes relaxed specificity of taq DNA polymerase. It could be possibly a reason too. I am also suspicious you are obtaining a thick primer band at the end of the gel due to use of 10 times higher primer. primer concentration for taq DNA polymerase is 1 picomole per microlitre or 1 microlitre from 10 micromolar stock for a 25 microlitre reaction. if it is a regular taq, and a AT=GC amplicon, the reaction could give cleaner results in 200 micromolar dNTP and 1,5mM MgCl2.

The smearing of your ladder tells me that you have a problem with your gel conditions. I've had smearing like that when the gel rig (or combs) were not clean. Make fresh running buffer, wash ALL of the gel rig parts (comb, casting try, gel box), make sure your agarose is completely solidified before you remove your combs, and try running your gel again. The smear can also be due to loading too much PCR product into your gel. How much PCR reaction did you load into each well?

As Alireza, Simmi and Katie mentioned, it seems that the ladder is distorted too. looks like there is a problem with the gel preparation or electrophoresis conditions.

are you using the same TAE or TBE concentration for the gel and the running buffer?

and what's the power supply voltage?

As you still have the same problem with the ladder, I think the problem is not related to the reaction conditions and/or components. I think the gel is not homogeneous and it tends to be granular! Either it did not melt well, or there were bubbles! The gel must be well melted and placed in the casting try at the right temperature.

Dear Burnette,

I load 2 microlitre loading dye and 2 microlitre pcr product in the gel. And I prepared 40 mililtre agarose gel for minigel tank and added 2 microlitre Gelred dye.

It sounds like you didn't load too much of the PCR product. The smearing of the ladder does indicate that there was something wrong with the gel electrophoresis conditions. My guesses are either that the comb and casting tray were not clean or that you didn't fully melt your agarose before pouring it into the casting tray.

Smearing in the gel samples can be because of the improper washing with the wash buffers but as the ladder is also showing smear then it may be some problem with the gel conditions.

this problem have three reason

1- if you don't make the step of adding RNAase during DNA extraction so after running you found this smear in this case it is RNA.

2-this smear may be denaturated DNA , but this smear also appear in ladder so i recommend to you,

3 - check the quality of agarose and also if you don't wait for proper solidification of your gel before running it can make this problem, and also you can decrease the percentage of agarose.

1. Check your PCR product on 2% agarose gel.

2. Try TBE running buffer.

2. You should check

i. pH of your running buffer that is most important factor.

ii. Time and Voltage

Sometimes when the comb is not cleaned properly or there has gel slightly polymerised in the wells, it also affects how well your samples run in the gel, hence why you might see the smears.

First of all try getting better resolution for the ladder. Prepare gel by properly melting agarose and pour it in casting tray before solidification. Also take care of the buffer running conditions only once this is solved you can address issues with your sample.

dilute DNA before PCR as smear may be due to high concentration of DNA during PCR, ensure that your gel should solidify enough and apply standard current 5v/cm for electrophoresis tank so that buffer will not heat and your band resolves properly.

You may try to change the running buffer and all other buffer that used.

Please, rinse the gel casting utensils with phenol-TE followed by ethanol then keep in hot air oven for 2 hrs at 100 degrees celcius, use duly decontaminated gel caster utensils to make gel, make gel with autoclaved buffer, Treat both the product and standard ladder with phenol and run gel with autoclaved running buffer. There are many things are not written in the text book but we can do it by ourselves. hope for the best.

Smearing due to PCR reaction condition is usually sorted out by reduction of DNA template and sometimes also reduction of MgCl2 an d primers or increasing the annealing temperatures as depending on the primer sequence.

There could be several reasons:

1. Try changing the percentage of agarose.

2. Use RNAse during isolation process.

3. Use TBE buffer.

4. Make fresh tank buffer and check its pH before using.

There are three possibilities:

1. Use 1.5% in place of 1% agarose gel

2. Run the gel at 50V - 80V not more than that

3. Ensure that your DNA is in intact condition - you may do that by running a dummy RT and see if the melt curve is fine or not

This is could be due to contaminated DNA sample by protein.

You have to check you DNA, using Agarose 1%

Try to make negative no-template control.

Greetings, the smear bands, could be from the quality of DNA or the PCR components so try check it

pl check the % of agarose

clean the casting tray

check the quality of the sample ie DNA