What is the negative control, the positive control and the reagent control in the agarose gel electrophoresis that is prepared for the identification of PCR products? Could a ladder of DNA be a positive control? Could the components of the PCR kit (buffer+ taq polymerase+ dNTPs+ MgCl2) be the reagent control?
A positive control is anticipated to yield successful results under specified conditions, testing elements like the master mix, MgCl2 amounts, primer annealing temperature, and extension times. If the positive control fails, it signals potential issues with annealing, extension times, temperatures, MgCl2, or master mix setup.
On the other hand, a negative control in PCR is designed not to produce amplicons. It usually lacks a template or includes only one primer. Two negative controls, each with only the forward or reverse primer, shouldn't show visible amplicons. Any visible bands may suggest contamination or opposing binding sites for the primers.
Briefly: a negative control isn't expected to work under your Protocol conditions where the positive control is expected to work and reveal the desired results.
DNA ladder should not be considered as a control because it is added during the electrophoresis step.
The most useful negative control has both primers but water instead of dna template. This should always fail to amplify but when it starts to produce a pcr product of the same size as your expected pcr product then it has detected contamination in a reagent or the environment and that will be a problem because all of your samples will probably have contamination as well
Thank you Ahmed Adel Gereisha for your answer.
according to your answer, what is the difference between the positive control and the sample? are they the same?
Dear Dr. Suzanne Jubair
The positive and negative controls are samples that are used to confirm the validity of the gel electrophoresis experiment.
A positive control is a sample that contains known fragment of DNA (preferably of the same size as the target amplicon) and will migrate in a specific way on the gel.
A negative control is a sample that contains no DNA. But instead contains the components of the PCR kit namely the PCR buffer and reagent water.
The reagent control is same as the negative control. If you see a band in the negative control, it generally means that there is some form of contamination. It could probably mean that there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipette, or the tip used during the PCR reaction.
DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. But you may also use the ladder as a positive control for agarose gel electrophoresis.
Also, the PCR positive and negative controls can be used as the positive and negative gel electrophoresis controls, respectively.
Regards,
Malcolm Nobre
Hello,
in all reactions, the difference between positive control and negative control is the result. In the negative control, you are sure that there is no reaction and in the positive control you are sure of having a result.
So, in the case of PCR, the negative control does not put the template DNA which allows you to amplify your target sequence and therefore there will be no reaction, which will allow you to be sure that what you are going to obtained as the amplification product is that of the target sequence which is found at the level of your template DNA.
For the positive control, if you do the PCR to amplify and obtain a large quantity of target DNA (and not to verify the presence of the target sequence) there is no positive control.
Dear Malcolm Nobre , thank you so much for your comprehensive answer, I really appreciated it.
positive control could be a DNA sample that will get amplified and gets you the positive desired result in your protocol Suzanne Jubair .
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