While doing amplification with
Primers ( which already gave good amplification in same genus), it fails many time, eventhough the extraction has enough amont of DNA.
Inhibitors come in many types...some remove Mg from the reaction mix and the pcr enzymes need MG so the reaction fails. Many plant dna inhibitors have a structure rather like dna and absorb in the UV so give you a wrong idea of how much dna is really present. To get amplification you can use the ctab method of dna preparation which is good at removing inhibitors and use less starting naterial so the dna is purer. Alternatively phenol /chloroform removes most inhibitors Try amplifying dilutions of your dna...often the inhibitors can be diluted away and the smaller amount of dna amplifies well so try 1/2,1/4,1/8 dilutions of your dna and see if any dilution works, Whole genome amplification of the template dna works as the enzyme is less fussy about inhibitors then a pcr. The attached paper is informative
Inhibitors come in many types...some remove Mg from the reaction mix and the pcr enzymes need MG so the reaction fails. Many plant dna inhibitors have a structure rather like dna and absorb in the UV so give you a wrong idea of how much dna is really present. To get amplification you can use the ctab method of dna preparation which is good at removing inhibitors and use less starting naterial so the dna is purer. Alternatively phenol /chloroform removes most inhibitors Try amplifying dilutions of your dna...often the inhibitors can be diluted away and the smaller amount of dna amplifies well so try 1/2,1/4,1/8 dilutions of your dna and see if any dilution works, Whole genome amplification of the template dna works as the enzyme is less fussy about inhibitors then a pcr. The attached paper is informative
I agrée with Paul´s recommendations. As a first thing to try, I would set up reactions with serial dilutions of the template DNA.
One of the most annoying chemicals in plants are polyphenols. When extracted DNA pellet and solution are coloured in yellow/brown, that is in the most cases polyphenols. Besides inhibition of PCR they reduce overall quality of DNA by degrading it, and they cannot be easily removed from DNA as they form a strong complex with it. The way to avoid polyphenols is to pay attention to compounds of extraction buffer (like in CTAB method, mentioned by Paul Rutland) with anti-oxidative action (PVP, 2-mercaptoethanol, ascorbic acid) and increase their concentration, if default amounts seem to be not sufficient. In my experience, DNA extraction in cold conditions also reduces a damage to DNA.
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