I have a DNA sample and I don't know is it good or bad, I will do PCR for it.
I want to be sure that the bands that will appear after PCR are the wanted amplicon and not a primer-dimer. I need a positive and negative control in my PCR but I don't know how to make these controls in PCR.
Any help would be gratefully received.
Dear Noha Said
Positive controls could be obtained from lab culture identified previously,
Another form is to sequencing the products obtained in PCR
And other less expensive is using restriction enzymes than cut specific regions of pcr product into fragments that could be visualized into electrophoresis gel.
Usually negative controls are pcr mix + sterilized water in substitution of DNA
Hi,
Negative control is PCR reaction without DNA you just add water instead of DNA, positive control is from reference thing (Differ according to your work may be bacteria, virus, fungus ...etc) or if you have any positive sample you may just make sequencing after that you consider this sample as positive control. in diagnostic laboratory, positive control is prepared by cloning of PCR products.
Hi Noha,
To be able to choose the negative and positive controls you really need to understand what gene do you want to amplify. I would run 1 positive control and 2 negative controls.
For example, I want to see if my isolates are Pseudomonas, I use Ps. specific primer. My positive control would be P. fluorescent (which had 16s rRNA sequenced or you know for sure that it is P. fluorescent ).
And I would you 2 types of negative controls: E. coli and DI water as templates. Using E. coli, which is not a Pseudomonas species, as template can give me the information that the primer is specific for Pseudomonas and other species will not get amplification. Using DI as template to make sure that without physical DNA input, we have no amplification. This helps us prove that our techniques possibly good enough that we did not contaminate the reaction by taking DNA from environment rather than just interested isolates alone.
After running the reaction for a while, you can just run neg. control with DI since you can be certain about the specificity of primer.
And if you are worry about primer dimer, they are normally ~50 bp, and it would be toward the end of the gel, if the size of amplicon is very small, you should consider using low melting point agar for better separation. If the size is much bigger than 50 (>200), then you should not worry about that.
All the best.
Thanh Nguyen
1. Use 3 PCR tubes to explain:
1. tube 1: water+ all PCR components (negative control)
2. tube 2: your DNA + all PCR components
3. tube 3: a DNA template + all PCR components (positive control; you know this DNA template will definitely be amplified a target fragment you want, do you have this kind DNA sample?)
2. If your intended target PCR size is 500 bp., when you run a gel after PCR, you will expect to see: tube 1: no band on gel, tube 2: 500-bp band on gel, and tube 3: 500-bp band on the gel.
You can test your amplicon using restriction enzymes if it is possible to predict the sequence by blast among the public available databases. Subclone your amplicon and sent it for sequence, then blast it in ncbi and see what you got,
Hi Noha,
You've got excellent suggestions above. For negative and positive controls for your PCR, include tubes with no DNA and with known good DNA, respectively.
Also to begin with I would follow Azza's recommendation and run the DNA you're trying to test its quality on a gel with DNA marker and check if you can see a band of the expected size.
N.B. Primer dimers usually are small, so they ran fast and appear at very low molecular weight.
Good luck
If you get only one band and to be in the safe side, you may do sequencing for either the band itself (after gel extraction and purification) or by using the PCR product after its purification too. Most of journals/reviewers are now requesting to validate your amplicon size with sequencing and BLAST analysis.
At the first you must use a DNA marker for gel electrophoresis to know proper band or primer-dimer.
As mentioned above, negative control is a tube with all PCR components except DNA template and positive control is a tube with PCR product and known template that you sure it work. as a whole you can sequence your PCR product for final confirmation.
Hi Noha,
You may need to do additional literature searches on previous works that used the same primers in order to determine what band sizes to expect. Secondly use a molecular weight ladder that contains this band size in its range. I think your questions about positive and negative controls have been correctly answered above.
Good luck!
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