A simple method to prepare of DNA template suitable for PCR amplification from filamentous fungi will be valuable for improving experimental efficiency.Here,a method was developed which just needed ultrasonic treatmen...A simple method to prepare of DNA template suitable for PCR amplification from filamentous fungi will be valuable for improving experimental efficiency.Here,a method was developed which just needed ultrasonic treatment of the mycelium at usual condition,and the produced solution could directly be used as DNA template for internal transcribed spacer(ITS)amplification successfully.The PCR could be improved by additional treatment of 60℃water baths,but was not centrifugation.When the template amount was 0.5-2μL and the ultrasonic time was 7-11 min,there was no distinctly influences on PCR.The method was commonly used for M.purpureus,I.cicadae,Lentinula sp.,Flammul sp.and Dictyophora sp.etc.to detect target sequences of ITS,hygromycin resistance gene(Hyg),CRISPR-associated protein 9(Cas9),Citrinin gene C(CtnC),Citrinin gene D(CtnD),large subunit rRNA gene(NL),and so on.The method could provide a simple,rapid,safe and economic approach to prepare the DNA template for large-scale PCR of the special filamentous fungi materials.展开更多
基金Supported by the National Natural Science Foundation of China(81960692)the Science and Technology Support Program of Guizhou Province(2019-2776)。
文摘A simple method to prepare of DNA template suitable for PCR amplification from filamentous fungi will be valuable for improving experimental efficiency.Here,a method was developed which just needed ultrasonic treatment of the mycelium at usual condition,and the produced solution could directly be used as DNA template for internal transcribed spacer(ITS)amplification successfully.The PCR could be improved by additional treatment of 60℃water baths,but was not centrifugation.When the template amount was 0.5-2μL and the ultrasonic time was 7-11 min,there was no distinctly influences on PCR.The method was commonly used for M.purpureus,I.cicadae,Lentinula sp.,Flammul sp.and Dictyophora sp.etc.to detect target sequences of ITS,hygromycin resistance gene(Hyg),CRISPR-associated protein 9(Cas9),Citrinin gene C(CtnC),Citrinin gene D(CtnD),large subunit rRNA gene(NL),and so on.The method could provide a simple,rapid,safe and economic approach to prepare the DNA template for large-scale PCR of the special filamentous fungi materials.
