I am using genomic DNA from soil samples. I have sonicated the DNA template, blunt the end with enzymes, and then self-ligated them. Then I proceeded with PCR but the result is that my products smeared and no band was obtained. What could be the possible reason?
P/S:
- the DNA concentration is 70ug/mL and I used 2uL for each PCR reaction
- when I try with RE digested samples, multiple bands appear
Hi Yen,
Smearing of PCR product in gel run is probably one of the most discussed and answered thread in RG where I myself and other experts have contributed and commented in thousands of different occasions. Before posting any common question I would suggest investing little effort to stay updated.
I am sharing some parallel links relevant to your question.
https://www.researchgate.net/post/Smearing_in_agarose_gel_of_PCR_product2
https://www.researchgate.net/post/PCR_product_with_smear2
https://www.researchgate.net/post/Can_someone_help_me_regarding_a_smear_in_PCR_product
https://www.researchgate.net/post/PCR_smear
https://www.researchgate.net/post/Why_am_I_getting_smearing_after_PCR
https://www.researchgate.net/post/Why_am_I_getting_a_smear_in_PCR_results
https://www.researchgate.net/post/Why_do_I_have_smears_in_my_PCR_Plant_positive_control_and_negative_control
https://www.researchgate.net/post/PCR_product_shows_a_smear_in_the_gel
Best-
Touch down PCR or gradient PCR should solve your problem. Make sure the primers are specific and right if you have designed. But if you have followed primers from papers of established researchers you need not worry about primers.
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