Hi!

Simply your PCR did not work. What you are thinking is not at all band, some high molecular weight junk sitting in the well. Previously few people got same problem if you have patience you can go to the topics what you mentioned and search, your doubt will be cleared.

You told previously you got amplification, just make out what could be the difference! Is it because of new one what you used, did you follow the instructions carefully? Why you removed the marker, we would have got some idea by seeing that, is it some thing wrong with the gel or gel run!!!

If it worked earlier certainly some thing wrong went on, try to figure out that and repeat carefully, you get nice result.

Good Luck!!

Dear Lorenz

It is some high molecular weight entity ( may be genomic DNA). You need to be sure that while setting up PCR there are no contamination issues. Prepare reagents at one place and set up PCR at other place. Use freshly opened reagents fro your PCR.

Good Luck

Try to purify your PCR product with Phenol/Chloroform extracting, and try again. This will remove the protein or other things that block your wells.

If this doesn't work, try to digest your products with some restriction enzymes to check if you get some oversized DNA fragments by some reason.

Try increasing the concentration of gel run and lower the voltage to extend the run time

I agree that the band doesn't correspond to your PCR product it looks more like genomic DNA from your template. If you are seeing that much of a template after PCR that means you used too much template and that might interfere with your PCR reaction. I would try lowering the template,

Generally PCR needs 10 nano grams of DNA per reaction. If you take more DNA, in case PCR reaction fails the DNA degrades (95 C, Ta, 72 C for 35 times) and appears as smear.

what i think is the DNA is contaminated with protein and u first try to purify the DNA with Ethanol precipiation method. try to run the DNA after doing this technique, i hope u ll get good results.

Good Luck

I have the same problem with CloneAmp HiFi.  I don't have genomic DNA contamination because I use a gel purified vector as my template.  When I PCR purify my run, I get a large smear.  I am about to try using all new reagents because I am thinking there is some type of contamination with my tips or pipettes, but when I do PCR with other primers and template, I have no problems.  I made all new template and will try again with new filter tips and cleaned pipettes.  I'll let  you know if it worked!  

Oh, and try a gradient!

why DNA remain in the some gel well?

Hi!

I already explained that is not DNA remaining in the wells. It is high molecular weight junk. See in some wells though that junk was there still you got the amplification because of your DNA. Generally while extracting DNA especially during phase separation thinking we loose DNA we try to take up maximum supernatant, that was the reason. If we do carefully we do not get this problem. Generally it happens that the junk appears where ever it is there.. Just check my DNA gel image some of the samples are clean some are having junk. But when I did amplification all of them worked. without any problem.

I guess this is a full blown support group so I'll share my private art piece - I call "lazy bands"..

In case of long PCR, you need to decrease the concentration of the starting DNA (particularly if you are amplifying a whole plasmid), because their is high risk of self priming

📷

Hi, in my case I was re-using the gel, I prepared again and used everything new and the stuck go away. There was some smear but in a less degree.

Hi, I have the same problem with this kit. I´m trying to add some sequenc to my DNA of interest. Firstly I amplifie DNA of interest form cDNA and than when I do rePCR, I allways see smears on gel. Did you find any solution?

Gil Zimran I'm having the same problem when performing the mutation procotol from QuickChange, did you finally solve this or at least find out what's happening?

Ramiro Illanes Vicioso , It was a long time ago so I don't remember anything. seems like other people in this correspondence are on top of this. Generally I'v abandoned the QC mutagenesis kit because it's expensive and not so effective. Now I'm just using mutagenic primers and amplifying fragments for Gibson assembly - consider it..

I everyone. I am having the same problem but my samples are RNA. I have treated all my samples with DNase so I believe that it is not DNA that is stuck in the wells.

Do you have any ideas? Something with the resuspension of my RNA pellets or related with EtOH washing?

I send a picture attached.

Thank you very much.