I am planning to standardise a qpCR assay for gram negative bacteria. DNA extracted by boiling lysis method works fine for conventional PCR but, will it hamper qPCR assay as it is very sensitive? So can we employ DNA extracted from gram negative bacteria by boiling lysis method for qPCR analysis?
Dear Yugendran,
I don't thing that you can use boiling lysis for qPCR analysis, with this method you're co-extracting DNases and other inhibitors/contaminants, that will affect your results.
Have you tried to use an old extraction for PCR? ... it works? .. I had good results, for recently prepared DNA by the boiling method, but when you store this extraction and do more than two freeze-thaw cycles, your DNA prep is not longer giving the same nice results.
I will suggest to use a more clean DNA extraction protocol if you're not able to use a kit.
hope this helps,
Beatriz
Dear Beatriz,
Thanks for your answer. I will be following kit method for template DNA preparation.
Dear Yugendran,
I think that you might find the phenol-chloroform extraction to give you better quality DNA, that can be used for qPCR.
Maybe it is something you can consider as it will be cheaper than purchasing extraction kits.
I have found that the boiling method works fine for end-point PCR.
Hope this information helps you.
Regards,
Dear yugender, for qPCR first we need total RNA and then make the cDNA from that RNA. so the boiling method is not suitable for RNA extraction as RNA can degrade
Dear Sooraj Bajinath,
Thanks for your reply. I will definitely try the phenol-chloroform extraction.
Thanks
Jawed sir,
How are you kaise hain aap? Where are you working now?
Sir I was talking about qPCR assay for DNA amplification. For RNA I am following kit method.
Thanks
Hi, Most of researchers we prefer using boiling method especially for PCR amplification of chromosomally-encoded genes. The method is cheap, faster, and effective as impurities are greatly reduced.
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