黄瓜DNA提取及优化SSR反应体系的建立

Extraction of Cucumber Genomic DNA and Establishment of Optimal SSR Proccedure

本文简化了黄瓜DNA提取程序并探讨了黄瓜SSR反应程序及体系。反应体系25μL:10×buffer 2.5μL,2.5mmol/L MgCl_2 2.0μL,2mmol/L dNTP2.5μL,gDNA模板(10ng/μL)2μL,forward primer(10pmol/μL) 0.5μL,reverse primer(10pmol/μL)0.5μL,ddH_2O 14μL,Taq DNA聚合酶(1 U/μL)1μL。反应程序:95℃预变性5 min,94℃变性30 sec,49℃退火1min,72℃延伸90 sec,循环次数35,72℃延伸5 min。

Abstract In this paper we simplified the DNA extraction procedures of cucumber and optimaized the SSR-PCR reaction system. The SSR-PCR reaction was optimized in 25 μL mixes containing 2 μL ofgenomic DNA （10 ng/μL）, 2.5 μL MgCl2 （25 mmol/L）, 2.5 μLdNTP mixtures （2 mmol/L）, 2.5 μL 10× PCR buffer, 0.5 μL forward SSR primer （10 pmol/μL）, 0.5 μL reverse SSR primer （2 0μmol/L）, 1 μL Taq polymerase enzyme （1units/μL）, and then added 14 μL ddH2O to the total volume. The amplification temperature profiles were as following： 5 min at 95℃ for pre-denaturing, followed by 35 cycles of 30 see at 95℃ for denaturing, 30 see at 49℃ for annealing, 90 see at 72℃ for extending, and finally 5 min at 72℃.