Research on the Populations Variation of Alnus cremastogyneI. DNA Extracting and Protocol Optimum for PCR

Taking China's endemic tree species Alnus cremastogyne and other 5 Alnus species as test materials, the DNA extraction of genome and the amplification of PCR were studied. Two methods were tested, i.e. freezing treatment+5% CTAB and liquid nityrogen+5% CTAB. It is proved that the optimum PCR program is as follows: pre denaturating under 94 ℃ for 3 min., denaturating under 94 ℃ for 18 seconds, annealing under 36 ℃ for 80 seconds and extending under 72 ℃ for 120 seconds. After 40 cycles, the sample is reacted for 10 minutes under 72 ℃. PCR system includes buffer 2.5 μL, d NTP 2.5 μL, primer (s60+s155) 2 mmol·L -1, Mg2+ 3.0 mmol·L -1, Tap enzyme 1 U, DNA 40 mg, then adding ddH2O to 20 μL. This study may provide some references for the application of RAPD technique in genetic research of alder tree species.