are you routinely running a no template control in your pcr and does it give a negative result ?

are you routinely running a no template control in your pcr and does it give a negative result ?

Paul is right, negative control will be so helpful in guiding your options! If you still have the main stock, make another diluted stock (working stock). Such problems could be raised from re-using the working stock (melting-freezing).

In 2 wells after marker you had very weak band but in my experience according to your wells are bright you had total dna(high con dna in your samples) so semears have same reason (but with lower con).

Did you check dna con?

If you had samples in freez maybe freez and thaw fragments your samples

reduce the number of PCR cycles, and amount of DNA polymerase and concentration of primers

Hi Paul, Thank you. Yes, I always run a no template control. In the figure, that will be the third sample (dH2O)

Thanks everyone. However, we thought the problem could arise from freeze-thawing or degradation of reagents so we purchased completely new sets of every reagent. W also tried reducing the cycle number from 40 to 30cycles but the situation did not improve.

Hi,

Use Phusion From NEB, very robust. Run the gel longer. Make fresh primer and template stock. Use correct % of gel. Lower percentage can give smear.

- Anand

there's a lot of primers left, was it the case before?

It seems like DNA degradation and/or contamination check it by negative control.Moreover try to do DNA check for your sample quality again using agarose gel if you notice this banding smear pattern again, be sure it is a degradation.

Be sure the rest of your samples is in a good storage condition.

Try to Work with aliquots; avoid freezing-thawing of your sample.

Hope the best for you.

How much DNA do you put per reaction? It would be helpful if you shared previous pictures from when you got OK results.

Emma,

There are some suggestions in this post: https://www.researchgate.net/post/Why_I_saw_smear_band_in_the_agarose_gel

I particularly like the suggestion of using less DNA template going into the PCR.

I would also add the following:

You should be able to get your DNA ladder to look more clear. If not, then the problem is likely with the Gel (e.g. how you make it) or the way you run the electrophoresis (voltage, buffer choice, etc).

Good luck!

-Rolf

Dear Emma,

It seems to be gDNA contamination but the DNA is not intact it is degrading. You may run a positive and a negative control for the gel. In positive control add your cDNA or gDNA and in negative control all the reagents except template. Now you can run the gel and see what is the issue.

Anush

It seems like your gel of electrophorese and buffer tunk is not made well because the ladder has smear as well.

the first one is ladder, isn't it?

Try to exclude contamination....Also, as your ladder is smearing as well, try to take care of your gel electrophoresis in terms of the buffer, the tank,..etc.

Hello Emma,

what I'd do is:

- Check/change your TBE buffer. Are you using the right concentration? 10X --> 1X

- Decrease the concentration of your gDNA.

- Seems like its the result of contamination but please do include your negative control. You may ask someone in your lab to load your samples on the gel (different hands, different results) to double check contamination or what it is?

- Decrease the cycle or play with temperatures.

- Decrease voltage. If you run at really high voltage, it will always give you smears at the very first beginning of the run.

hope these will be helpful,

cheers.

Hi Emma

Here are some factors such as:

1- Decrease the concentration of MgCl2 it's one from reasons to give non specific bands.

2- Decrease the concentration of your gDNA

3- Decrease the number of PCR cycles

4- increase the annealing temperature

Good luck

It looks like the template was contaminated with genomic DNA.

Thanks everyone. I will try to address the issue of contamination as the initial troubleshooting as kindly suggested by many researchers.

I would try;

- Use new buffer (TAE/TBE) in the electrophoresis tank and use the the same buffer to make the gel.

- Try a lessor percentage gel (if viable).

- Run the gel for longer (i.e. after taking this earlier picture, carefully place the gel back in the tank and run for another, say, 20-30 mins). Even in the smeared wells, there seems to be bands at the 2nd marker from the top. Maybe the gel needs more time to resolve.

Good luck.

Dear Niba

What is the original sample used for DNA extraction? Does it fresh or frozen blood. If you use frozen blood, a specific protocol differ from that used for fresh blood. This is mainly due to while the target of DNA extraction is WBCs, freezing and thawing cause rupturing of RBCs and release of observable haemoglobin which interact with proteinase K. Regard

hello Emma Firstly check The quality of DNA. Try another methods of extraction

may be elongation of salt step will cause damage of genomic DNA

We are using dried blood spot as our sample for DNA extractio.