There are many factors that can contribute to smearing: from DNA degradation, non-specific primer binding (have to check primer specificity and optimization), non-optimal temperature (have to do optimization), too much MgCl, and to many others. Most commercial mastermix should be okay for all routine PCR, but you may want to check the content breakdown, or just switch to another brand. Worst come to worst, it is your DNA sample has already degraded, meaning you need to resample.
Hi Oadi,
If this is the very first attempt for PCR...then it is advisable to optimize the reaction initially with 1-2 samples, to get clean results.
Optimize for annealing temperature, MgCl2, Tempelate amount, eleongation time etc. and see what is the best condition for your expected amplicon size. Then go ahead with th remaining samples....
bye
The cause of smear:
1. High quntity of DNA template
2. Degradation of DNA
3. Degradation of DNA polymerase
4. loading of large amount of PCR product
5. Contamination of DNA by protein
many reasons contribute to this like :
1. Tm of primers( do gradient PCR to know accurate Tm of your primers)
2. do not use to much DNA templete
3.do not use MgCl2 more than 3mM final concentration.
4. primers used should me 10uM stock
one very small question. U r adding buffer when you are making gel?? right
because your marker seems to be smeared too
good luck
Thank you for all yours comments and suggestions, I found the problem: The Master Mix was not good brand, just i switched of with another master mix and smear gone.
Thank for every one.
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