5 ul DNA in your reaction volume and 43 PCR cycles is a lot. I often get smears when i use so much DNA of so many PCR cycles. Try loading less DNA or use fewer PCR cycles.

5 ul DNA in your reaction volume and 43 PCR cycles is a lot. I often get smears when i use so much DNA of so many PCR cycles. Try loading less DNA or use fewer PCR cycles.

you do not say how much dna is in 5ul but I agree with Pim Dröge it is probably too much dna and so too much pcr inhibitors present. Measure the dna and use about 50ng and run between 25 and 35 cycles ( prepare 4 tubes and remove at varying cycle numbers) . there are 2 main possibilities,,,,too much pcr inhibitor so no good amplification or amplification is too good and there is too much product and it is smearing and in the latter case lower cycle numbers will clarify this

I agree with Dear Dr Paul Rutland and Dear Dr Pim Dröge . In addition to what doctors have said, I think the time of the denaturation that you applied: (10 seconds) is so short and the same thing to 20 seconds for the annealing and the extension is 20 seconds. If you try in longer time, like 30 seconds, I think it will be more efficient for a good PCR product.

I suggest that you check the quality and conc. of your DNA (e.g. using NanoDrop) before using it as a template for the PCR. You also better use a positive control if it was available to make sure things went well in the PCR.

Regards

Lot of inhibitors and nucleases contamination, check first negative and positive control using fresh reagents and Mannaa suggestion considered. Cycle number 32-35 is ok, DNA concentration per reaction based on DNA quality and quantity. what's you detection limit of Primers?, its also matter. Check Detection limit of primers, quality and quantity of DNA, First check with positive and negative control.

Best@

all is right, additionally try to take (2-5 ul of your DNA) and run on gel , mix with nuclease free water if it is e-gel, and then detect its integrity, if you have fragmented DNA or primers you will get such picture, Good Luck

The PCR cycle number is compared to DNA volume is bit high. Try to minimize either DNA volume or number of cycles. And also try low voltage value with long time period. That may be facilitate DNA bands separation even better.

Looking at the gel pic I could make out that the DNA is sheared and the amount used for PCR is little high. So dilute the DNA and run the PCR

DNA ladder to the right not straight. At what voltage was that electrophoresis run at.

That could be a factor too.

Check the quality and quantity of DNA in the PCR reaction. Also use proper control

Paul Rutland and Pim Dröge both have good recommendations. One thing I can add is I like to have at my disposal is an inhibition control assay. Take you DNA sample in question and mix it 1:1 with a PCR primer pair and template that you know works wells. In a separate rxn add water 1:1 with the inhibition assay. Not a bad idea to also make a dilution of your DNA in question then mix 1:1 with the inhibition assay. I use qPCR for the sensitivity but end-point works too as long as you keep the cycle # down low. Compare the control water:inhibition control and your DNA:inhibition control.

I use a gBlock with a short DNA segment from a plant photosystem gene with no homology to anything I work on. gBlocks from IDT are cheap and will last years and years.

Jeffrey

You should recheck the following:

1. Volume and Conc of primers and MgCl2 added to your PCR mix

2. dNTPs if they are still working.

3. Thermocycler is working condition because seems nothing is amplifying

4. Visualization dye

5. Consider apply 2.5% DMSO

6. Prepare fresh running buffer (TAE or TBE)

7. The electrophosis in working condition?

Finally what other people said check the quality of your DNA. It will be a complete troubleshooting to find the problem.

Best wishes,

Due to many reasons

-contamination with lysis enzymes or low DNA concentration or low quality of reagents or stains used

Your sample is degraded due to contamination

Must use positive control for that DNA sample,

It seems you didn't get specific bands as the image is negative for products of desired length/size (253 bp). That means either your sample is degraded or primer didn't anneal which may be attributed to a number of factors like overloaded DNA, improper DNA dilution, gel quality and so on. But, fitst u should use a positive control to investigate the reason and find out more specifically

firstly check the quality of DNA and runs it seperately on agarose gel electrophoresis to detect if DNA degredation present.

hi

it is very important that you have to check the quality of dna extraction protocol

in my opinion,your DNA extraction protocol was not correct.(please check the buffers and reagents)

1.you should run your extracted DNA on agarose gel electrophoresis to check the your DNA before PCR reaction.

2.another point is using internal control for your PCR.

3.decrease number of your cycles

4.check the concentration of mgcl2

5.increase the annealing temperature for prevent from non-specific binding of primers

best

Quality of DNA should check.

Hi Vasilli

I think you check this reference

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=13&cad=rja&uact=8&ved=2ahUKEwje_ITqvtzfAhXG3SwKHQRvCAIQFjAMegQIAxAC&url=http%3A%2F%2Fpalumbi.stanford.edu%2FSimpleFoolsMaster.pdf&usg=AOvVaw3gkOipwU7ZNRnEnxVULe2m

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=17&cad=rja&uact=8&ved=2ahUKEwit3cuev9zfAhUGjywKHZsTC9IQFjAQegQIARAB&url=http%3A%2F%2Fwww.protocol-online.org%2Fbiology-forums%2Fposts%2F12073.html&usg=AOvVaw3HM_oE7Y9OVMJutVkdlqY2

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=18&cad=rja&uact=8&ved=2ahUKEwit3cuev9zfAhUGjywKHZsTC9IQFjARegQIBBAB&url=https%3A%2F%2Fwww.scientistsolutions.com%2Fforum%2Fdna-pcr-real-time-pcr-standard-polymerase-chain-reaction-pcr%2Fsmear-problemhave-picture-gel&usg=AOvVaw0uZrBjRFWX_pN0GSPb8xiH

In my opinion, 100 volts for electrophoresis is very high. You must also setup your pcr program.

First of all you have not mentioned the concentration of DNA in your 5 ul, If is around 50 ng/ul. I would suggest you to use less amount may be around 2 ul. We also need to reduce the quantity of primers to 1 ul as 4 ul of primer is very high concentration.

First make sure that the material used and the quantities used in the experiment@Vasilli Michael Kasimov

Hi Vasilli Michael Kasimov

- you can use 1ul of DNA, 1ul from each specific primers

- PCR protocol modification :

Initial PCR activation: 94 Degrees Celsius for 3 mins

30 Cycles of:

- Denaturation: 94 Degrees Celsius for 10 seconds

- Annealing: 55 Degrees Celsius for 1 min

- Extension: 72 Degrees Celsius for 20 seconds

- Further Extension: 72 Degrees Celsius for 5 mins

Infinite Hold: 4 Degrees Celsius

Good luck

It could be a number of reasons.. your initial DNA concentration could be too high or, as you're using genomic DNA as your template, your primers might be non-specific so that they are producing a lot of products of different sizes.. Also, 40+ cycles is a lot, usually 30-35 cycles is enough.. If you're worried about DNases you might add some DNAse inhibitor to your reaction (e.g. mercaptoethanol or EGTA)..

Are you including positive controls? Without the proper controls, it is difficult to troubleshoot which component(s) may be the problem. Note that all the components of PCR (e.g., polymerase, DNA/primers, dNTPs) can become (directly or indirectly) inhibitory at high concentrations .

Is possible try to test lower DNA concentration, lower number of cycles and gradiente in the temperature annealing.

I often get smears when i use so much DNA of so many PCR cycles .