Two PCR mixtures, 55 and 60 micro litre total volume. The mixture contains 10 and 15 micro litre of DNA template respectively. PCR for Panfungal genome targeting ITS was performed. Reaction was picked up only in tube added 10 micro litre of sample(DNA template) and not in 15. Why???
Hi Kathiravan,
The most likely explanation would be the presence of a PCR inhibitory substance in the DNA preparation. In addition to contaminants that may have been carried through purification the presence of EDTA in the buffer used to elute/store the DNA can bind divalent cations and interfere with PCR if at high enough levels. Another, less likely possibility is that there is some dilution of other reaction constituents in the larger volume reaction.
Good Luck,
Frank
Kathiravan Vayanaperumal
Regarding your question, Frank R Burns 's answer about the inhibition in PCR reaction is correct but your understanding about PCR looks a bit nascent.
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