i've done sensitivity test for primer with consentration of sampel 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, and 15%. and the result of electrophoresis show 5% band is thicker than 10% and 15%. is it correlate with masking DNA ?
thank you before
Hi there
if you are saying that lower concentrations of DNA correlate with higher yields of PCR amplicon then no this is not a masking effect; Rather an inhibitor effect. At low concentrations of DNA with inhibitors there might be sufficient moles of target sequence present to obtain amplification but an insufficient amount of inhibitor(s) to inhibit your reaction. At higher amounts of DNA the concentration of inhibitor can exceed a threshold and actually start to take effect so you get manifest inhibition and hence lower amounts of your PCR product
Inhibitors that exert an effect above a certain concentration or threshold but not below that concentration are numerous and apart from blood derived DNA where things like Heme can exert inhibition are (for most other types of samples) salts that have bled through into the final purified DNA. In particular DNA purification kits contain guanidinium salts that are designed to denature proteins facilitating purification and providing a stable extraction environment:
If these salts bleed through into your final purified DNA they inhibit downstream enzymes/proteins particularly at higher concentrations of purified sample
Poor quality DNA resulting in the potential for such inhibition is the result of incomplete removal of such salts and this in turn is indicated by a 260/230 ratio of less than 1.0. Incomplete salt removal can be countered by (for example) leaving your ethanol salt based wash buffer on the column for 1 minute before spinning through; and then repeating this desalt wash
This invariably leads to 260/230 ratios > 1.0 and minimises the sort of inhibitory effects described
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