菊属植物RAPD反应体系的建立

在ＲＡＰＤ反应中，有许多因素影响结果的稳定性和准确性．该文选取了菊属６种植物，对ＲＡＰＤ各种反应条件进行了摸索．结果表明：菊属植物较为理想的反应体系为：２０μｌ反应体积含１０ｍｍｏｌ／ｌＴｒｉｓ－ＨＣｌ（ｐＨ为８．３），５０ｍｍｏｌ／ｌＫＣｌ，２ｍｍｏｌ／ｌＭｇＣｌ２，０．００１％ｇｅｌａｔｉｎ，２００μｍｏｌ／ｌｄＮＴＰ，５０～２００ｎｇ引物，０．７５～１．０单位（Ｕ）ＴａｑＤＮＡｐｏｌｙｍｅｒａｓｅ，２～１０ｎｇ基因组ＤＮＡ．在２０个核苷酸引物情况下，反应条件为：９２℃、３ｍｉｎ预变性；９２℃、４５ｓ，５０℃、１ｍｉｎ，７２℃、２ｍｉｎ，循环４０周；最后一次延伸为７２℃、１０ｍｉｎ．应用上述反应体系进行扩增反应，反应产物在２％琼脂糖凝胶上以５Ｖ／ｃｍ电场强度电泳２～４ｈ，获得了满意的ＲＡＰＤ图谱

Selection of RAPD Reaction Conditions for Genus Dendranthema

Many factors of the RAPD reaction procedure may influence the results. This paper presents selected RAPD reaction condition for analysis of genetic relationship among Dendranthema species. A more extensive study of influential factors is also presented. To get good results, the reaction should be performed in a volume of 20μl containing 10mmol/lTris-HCl(pH8. 3), KCI 50mmol/l, MgCl2 2mmol/l, gelatin 0. 001%, dNTP 200μmol/l,primer 50￣200ng, Taq DNA polymerase 0. 75￣1. 0 unit, genomic DNA 2￣10ng. The mixture was laid on thermal cycle. After 3 minutes at 92C, 40 cycles were run. Each cycle consisted of 45 seconds at 92℃, 1 minute at 50℃, 2 minutes at 72℃. Then 10 minutes at 72℃.Plant growth state, methods of DNA isolation were also discussed as influential factors.