Hello. In our laboratory, we are currently conducting Loop-Mediated Isothermal Amplification (LAMP) to detect insert DNA in plasmid vectors.
When using the LAMP method, we typically use 8 tubes, where 7 tubes contain the template in either the same concentration or serial dilutions, and the last tube contains D.W instead of DNA as a negative control (N.C).
Recently, we attempted to use the Thermo 7500 PCR system to detect inserts to compare it with other analytical equipment, and I encountered some confusion.
According to my professor's advice, we used the Quantitation-Comparative Ct (ddCt) method among the three experimental types available in the 7500 system (Quantitation-Standard Curve, Quantitation-Relative Standard Curve, Quantitation-Comparative Ct (ddCt)).
While assigning wells, there is a setting to choose which well to use as the reference sample. Is it correct to use N.C as the reference sample in this experiment?
We currently do not have a reference sample such as a housekeeping gene or primers for it in our lab. The concept of an endogenous sample also needs to be clear, making this situation very confusing. In this case, what experimental method should we use, and is a reference sample essential?
I think you need to step back and clarify exactly what it is you're trying to determine, here.
Don't rely on in-built machine settings, because they're usually for general everyday scenarios rather than specific questions, and also because if they go wrong you're unlikely to spot it.
Are you trying to determine how many copies of your insert are in each plasmid? And if so, how are you preparing the plasmids?
Basically, a bit more information is necessary before we can even answer this question.
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