Dear Community members,
I have separated the bacterial cells in nuclease free water and used thermal lysis.
Apart from proteinase and cell debris, what are the potential inhibitors in pure cell lysate that will affect PCR.
Thanks in advance.
There are other inhibitors, but they should not effect the PCR. From personal experience, the best results are when the cells are added directly to the PCR mix the PCR is done straightaway, the start of the PCR provides the thermal lysis.
Hello Ismail,
PCR inhibitors abound in cell lysates. These inhibitors may act directly on the nucleic acid of interest or may be acting as inhibitors of enzymes involve in the amplification reaction. Aside from proteinases, Calcium ions, large carbohydrates, and proteins such as myoglobin and IgG are found to interfere the process, too.
With pure and inhibitor-free DNA and proper PCR protocol, certainly you will be successful.
Hope this helps.
Best,
Arizaldo
Sorry, I should have added, there was a similar question a few years ago
(www.researchgate.net/post/Can_anyone_share_a_good_protocol_for_direct_PCR_of_genomic_DNA_from_mammalian_cells_without_DNA_extraction)
Dear Steve and Arizaldo,
Thanks for your answers. I will try with direct PCR on the cells without DNA extraction and also I will compare the results with extracted DNA. Hope I will get better results.
Regards
ismail
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