Hi Muhammed

Smeared band problem usually comes from non optimized PCR protocols, although purity of DNA is  also a parameter in optimization. You must optimized annealing temperature of your primers very carefully. Performing gradient PCR for determining your primers Tm will be helpful. Secondly excessive concentration of dNTP, template or MgCl2 may cause unspecific bindings, which can be seen as smeared bands. You should check these parameters separately and optimize the concentrations if necessary.

http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full

You can check this paper out to find more solutions for your problem. 

Good luck

Basak

Too many cycles can also result in a smear even when all other conditions are optimal.  Set up 5 or more identical reactions and remove one at 10 cycles, 15 cycles, 20, 25, 30 cycles ... and see where the peak product is produced.  Or perhaps someone in your group does real-time PCR and you can monitor product production with that.

Dear Senturk, Foley and Gajda

Thanks for guiding, I shall try to seek the solution.

Can you write your protocol, products used (and concentrations) and primers sequences?

Hello,

It is said that touch-down PCR is applicable for up to 10 kbp amplification, smear band, etc.

here is the cycle:

1. 98 degree=2 min

2. 98 degree=10 sec

3. 74 degree (depending on primer tm)=60sec./ kb

4. 2-3 cycle for 5 times

5. 98 degree=10 sec

6. 72 degree (depending on primer tm)=60sec./ kb

7. 5-6 cycle for 5 times

8. 98 degree=10 sec

9. 70 degree (depending on primer tm)=60sec./ kb

10. 8-9 cycle for 5 times

11. 98 degree=10 sec

12. 68 degree (depending on primer tm)=60sec./ kb

13. 11-12 cycle for 15-30 times

14. 68 degree = 7 min

15. 4 degree = hold

denature 98 degree is for the template in case of being GC rich. the annealing+amplification temp is stepped down so that the primer specificity can be maximized. The Tm of the primer that I used was 65 degree and it worked well. But if you change the temperature of elongation, check the optimum elongation temperature of your polymerase.

Adjust the cycle with your experiment and if you have further question, feel free to ask.

Good luck!