PCR products stick in gel wells, PCR problem

From the image all i can say is that the DNA load is too much. you need to dilute the sample before loading and also dillute the templet DNA appropriately for the PCR.

Are you loading a PCR product? IF so, you need to dilute it more or add less prouct totthe wells. If you're loading genomic DNA load WAY less! If the PCR did not amplify you're running genomic DNA. TOO much DNA will prevent PCR amplification. Try to use NanoDrop or plate reader to quantitate your genomic DNA and PCR product.

I had the same problem several times. Looking at the picture I can say that is a lot of DNA. Defnately amplified! I would guess PCR is fine. Try diluting the sample and run the gel that sholud give you an idea where to fix it.

Hi everyone, I attached a gel here and take a look if you are interested in. Thanks, JH

add DMSO and NH4 positive ions will open your negative loaded DNA and let your primers access the GC rich "DNA spaghetti"
or simply buy MyTAQ that has it all in the buffer optimized better than any other TAQ supplier...

Thank you everyone for all the comments. They are very useful. Thanks for Jasmin,Siddharth, Michael, and Jorg's comments. The primers are stored in water, so as I think that is why it doesn't work properly. The problem must because the designs of this pair of primers. I will redesign the primers or just increase annealing Tm to 60 and increase annealing time.
Jasmin, the polymerase we use in our lab is Klen Taq, which is the only one we usually use for PCR amplification in this lab. It has its reaction buffer. the product length is not a main issue. It never worked when I use this pair of primers to amplify and to check the KnockIn gene (2400 bp PCR product) or the KnockOut gene (will have 800bp PCR product). However, these primers work properly when they were pair with other primer individually.
Manojkumar, the DNA loading dye is okay. Other PCR product work normal when using the loading dye. Tm is absolutely the main problem in my case.
It probably is better to redesign primers than keep trying all different PCR conditions.
Thank you guys for these great suggestions.
JH

Also, ideally a primer pair should have Tm's within at least 5 degrees of each other if possible.

A primer with a Tm of 43 degrees will not bind DNA at an annealing temp of 55 degrees. The annealing temp should be a few degrees lower than the Tm of the primer. Also, the template DNA could be increased to 50-100ng.

Why not try to optimize or adjust the conditions of your reaction.

Hi Jun, still in the lab?
Linda and Siddarth made a good suggestion. Jasmin´s comment was also important: don´t use water when storing primers for a longer time. As only your forward primer was newly synthesized and the reverse one is pretty old I would suggest to order a new reverse primer. Possibly you can choose primers with similar Tm´s on that occasion? Would be much better. What do you see if you apply a sample of your PCR reaction on a gel before doing the amplification? What happens if you try one or two different polymerases (like Phusion). We need your input now.

hey JH
the extension time is not an issue..and i wouldn't suggest using DMSO..you're just complicating your experiment...why not try decreasing the annealing temperature to 50 as one of your primers has a Tm of 45?
if that does not work i suggest you redesign one of your primers. add a few bases to the primer with Tm 45 or remove a few bases for the primer with Tm 60

Hi Jun,
you need to first optimize your annealing temperature by doing a gradient pcr (50 to 65C), the absence of product can indicate the need for a lower annealing temperature.and increase the annealing time.
As a general rule, extension times of one minute per kb should be used. A final extension of 5 minutes at 68°C is recommended.
good luck.

Hi Jun,
could you show me the amplicon sequence? What is the time of elongation step?
Increse the tempature of denaturation step (98° for 20''), alternatively, use a touch down PCR. (65° to 54°)
Good luck.

According to me i guess there are following possibilities

1) the gell loading buffer which you used for loading the sample is not proper.
if you have used he same gell loading buffer for the markers and your control then this possibilty is ruled out

2) PCR is not being carried properly.
this mght be because the designed primer pairs are not proper. Tm difference should not be more than 5 degree celcius. In your case the Tm of your primer is 45 degree and 60 degree but you have set the anneling temperature at 55 degree. In such case only the primer with the Tm 60 will anneal and not the primer of 45 degree. this will result in amplification of single strand.
But as in your case you are getting a bright band, this could be due to genomic DNA; indication negetive PCR amplification.

So i will suggest that check the gell loading buffer and redesign your primers with Tm difference not more 5 degree celcius.

If you want to check if your PCR product is single strand amplification then perform PCR in two sets. in one set add both the primer and in the another set add only the primer with Tm 60 degree. keep the condition same as you mentioned above. keep annealing temperature 55 degree for both the set. observe the result.