I got the desired band after pcr. I extracted DNA using boiling method. But few months later I am using the same DNA template and same pcr protocol is also maintained. But this time, I am not getting those bands which appeared before. I have re-cultured the sample on the agar plate in order to use fresh inoculum for making DNA template. All the pcr reagents are working well. I kept my primers and dna template at 4 degree and pcr reagents at -20 degree.
Go back to your primer original (undiluted) stock and prepare more primer diluted for pcr. Run a pcr with both old and new template at 4c lower annealing temperature than you optimised for earlier just to make sure of a (dirty) amplification if possible. Ideally borrow taq and pcr mix from another researcher in case something has happened to your reagents.Again ideally borrow a dna and primer set from someone else just to make sure of a positive amplification as a test of reagents except primer and template.Hopefully this will show which reagents are working and narrow the search for the problem component
Paul Rutland Would you please suggest what can be reason if your control s 16 gene is not producing the expected results in bacterial sample in q PCR? While in same reaction the another gene showed absolutely normal peaks or results..
What can we judge from this behaviour of s 16 gene ?Does it question experiment validation?
The convention in RG is generally one question generates a a thread of relevant answers Hira. It is better to submit this as a new question then different people with appropriate expertise are more likely to see and respond to your question
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