Should I use empty plasmid to be PCR negative control? And why?
Depends on the primer you designed.
If the plasmid contains any site that can be bound with primers, you will get a band in negative control.
Please note that the primers dont be necessary 100% to fit the sequence on your plasmid. It may just bind a few bases to start the extension phase.
Therefore, usually we add water instead of plasmid as -ve ctrl
Hello, Dr. Alex! Thank you for your answer. However, if I check annealing ability of primers and empty plasmid in Primer-BLAST, and the result is not annealing, should I still use that plasmid?
Primer-Blast may not indicate the real situation because the short oligonucleotide of primer may have mismatch tolerance and bind to several site.
Why dont you include both two negative control to see if any bands generate?
Dr Alex, I am sorry not to understand your meaning. Can you explain your suggestion clearer?
Thank you, Alex! Can you tell me how to check if mismatch tolerance happens?
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