Dear Colleagues,
I was using the amplicon I cloned as positive control; therefore it always gives brighter bands as against the samples under investigation. I used this plasmid for PCR optimization (Ta, no of cycles, etc). My question is that is it correct to use this plasmid or I should use a just normal positive sample as the positive control?
Hi there,
I guess your positive control is made just to show that the PCR mix and the set of primers are OK, right? So it is just a qualitative control (the intensity of the band obtained is not the prime issue). Now the question is: is the DNA template similar in the control and in the samples? For instance if the control uses a purified plasmid and the sample tested is a bacterial clone you can expect some differences between the 2 PCR reactions. Ideally and if possible control should be of the same nature as for the samples in order to prove that the full system is working OK (but it is no always feasable).
Thank you Dominique for the prompt response. The plasmid was purified from a cloned of positive sample from the same plant. That is to say the plasmid and the remaining samples are from the same plant species.
Hi again, if control and sample templates are obtained the same way, there is absolutely no problem.
Dear Muhammed
As I know any DNA control should be from the same target under study. If you used from the same DNA, its OK, but for brighter bands, It may be due to using high conc. of control.
I would use a positive control from the same origin as the sample to be tested (specially when dealing with field samples). However if the positive control is just to identify the band on a gel and confirm that the PCR experiment works, when optimizing your PCR reaction, the use of plasmid should not be a problem.
I agree with the above answers you can use the plasmid as a positive control to confirm the PCR reaction works or not. But the genomic DNA extracted from cultures are not the same as the purified plasmid. I suggest when you are doing any PCR optimization use the genomic DNA extracted from cultures. When you are testing any optimized PCR you can use the plasmid as a positive control. But you should be very careful the purified plasmid positive controls are highly concentrated and high risk of PCR cross contamination.
Hi, it seems that a fragment very similar to your own one will set up the PCR better. Because as you know the quality of the DNA (like pureness) and the length of it and even the percent of GC are important in PCR optimization.
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