The aim of my study was to evaluate the sensitivity the PCR assay for the in-house primers that I had designed. The concentration of the extracted DNA was 564.4 ng/uL with A260/A280 ratio of 1.97 and A260/A230 ratio of 1.15.
The DNA upon PCR amplification yielded single sharp intense band. However, when the DNA was serially diluted the template with concentration >26 ng/uL yielded multiple bands corresponding to approximately 200 bp in all the lower dilution template.
But when again the undiluted DNA was used as the template it yielded single band.
The water used for serially diluting stock template was the same PCR grade water that I had used for setting up PCR assays.
What could be the possible reason for this non specific amplification which I get only when the DNA is diluted?
Thanks
Yugendran
the low 260/230 suggests that there are contaminants present in the dna. If these interfere with pcr then I suggest that your pcr works better as you dilute the inhibitors and so much so that the primers are not perfectly designed and as you remove the inhibitors the primers anneal in the wrong place and the better amplification results in many bands. Conversely as the dna and also the inhibitors increase then only the proper amplimer is produced because it is the most efficiently amplified and the inhibition acts more effectively on the side products so probably reduces the amounts of all products but the weaker ones disappear an the main band just gets a bit weaker
What are you using to extract your template. with the 260/230 reads try and get hold of the new Nanodrop One system, it will actually tell you the name of the contaminants (phenol HCL guanidine) and remove their contribution to give you a real concentration Of your DNA. Also as you dilute your template to that level, the chance of primer dimers
manifesting increases greatly as the primers are more likely to come into contact with each other.
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