Hi,

With the details you provided trouble shooting is not really possible.

What is 1x primer concentration? What type of gel do you use and how do you visualise your DNA? What is your primer length, what are the melting temperatures for your primer? What polymerase are you using and what type of template are you trying to amplify?

I use Novel juice from DirecX for visualizing and use the primer concentration of 10 microM  with Tm of 66 and 61 degrees and then 1% agarose gel. I  use 5 unit per microL of Abnova Taq polymerase and try to amplify CO1.

Assuming this is all the information you have, the first step is to check whether your mastermix is active. You will get a clue just by looking at the gel. Is anything happening at all? Are you getting any nonspecific bands, primer dimers, etc? If you aren't sure, try running the gel again with an unamplified control. 

If you are getting some non-specific bands or primer-dimer, that means that your mastermix is active and it suggests that there is something wrong with your primers or template. 

If nothing is happening at all, your mastemix is inactive. That would most likely mean that the polymerase is dead, or you have left out an important component, like magnesium or dNTP's. 

The lack of primer bands might be due to the fact that Novel juice might not stain ssDNA.  If you can you should try staining with CyberGold or something similar. Also short primers as yours will be very smeary on a 1%gel. You might try a 2%gel. If you have doubt regarding your primers you can run a denaturing PAGE to see if you get a nice single band for the primers.

Next, the question is why you don't get any product.

Try a test PCR with other primers on a simple plasmid. If you get that to work your buffer, enzyme and dNTPs are fine.

What template are you using? ´Plasmid or genomic DNA? From what organism? Are you sure your primers match the sequence in your template?

Check the UV absorption of your template... does it look like clean DNA? How much template are you using? Too much can mess up your PCR.

Make sure you have the correct PCR program.

For a super safe one go well below your melting temp with the annealing temperature.. something like

1min 95 denaturing

1 min 50C annealing

1 min per 1kb product size 72C

30 cycles should do.

If you have introns be sure to include their size in the extension time calculation.

Hope that helps.

Dear Andrew and Peter,

Thank you for the insightful comments. Unfortunately I don't have access to any other stains. My extraction protocol was phenol chloroform based on Nishiguchi's protocol and I got quite visible DNA in my tubes. Also I have run gradient PCR yet I have had nothing. 

how could I check for other contamination like phenol?

As we say in Farsi "Sepasgozaram" meaning Thank you.

Please follow a kit method of DNA extraction and run the PCR. If you get appropriate band on gel, you will be sure that your manual DNA extraction method needs to be modified (such as phenol concentration). 

Also please dilute your DNA (manually extracted, that you have now) 1 in 4 and run the PCR. Never know, it could be excess of DNA in the PCR mix.

Following along Dr. Daldrop's suggestions; maybe ask one of your colleagues for a control DNA and test your Mmix and primers. I'm a big fan of mixing expts when troubleshooting. If you can get a control DNA that will amplify and your isolation will not then mix your isolated DNA in with the control and check to see if you have inhibitors carrying through that aren't apparent in UV Abs.

First of all test your  primer performance doing a simulate Pcr on the website  COSMOS using the link below:

- register first, then:

- add primer sequences  to the primer database and note the ID of forward and reverse primer,

- download the template from Genbank  or  just copy/past in the canvas the most probable genome to whom primers are suppost to anneal; write down the Id of the template

-use  Pcr Db , please read help on the top of each db

-altermatively send me primer sequence and the genome target;

- let me know wich agarose gel  (% , PH and thickness) you have used

This procedure can be helpful for you to understand