柿AFLP银染技术体系的建立

通过对各主要影响因素的研究,建立了适于柿AFLP分析的银染技术体系:酶切体系20μL总体积中含纯化后的DNA450ng,EcoRI和MseI各3U,37℃酶切4h;酶切完成后加入连接液,37℃连接10h(或过夜),然后65℃变性10min;连接产物稀释5倍用于预扩增,预扩增体系中EcoRI和MseI引物均不含选择性碱基;预扩增产物稀释5倍用于选择性扩增,选择性扩增体系中EcoRI和MseI引物均含3个选择性碱基,选择性扩增完成后,加入10μLLoadingbuffer,95℃变性10min,立即冰浴;取6 5μL变性后的选择性扩增产物在6%变性聚丙烯酰胺凝胶上电泳,电泳完毕后,对胶板进行固定、脱色、水洗、银染、冲洗、显影、定影及干燥等银染程序。

The establishment of AFLP silver-staining technique system for persimmon

After systematical study on the main factors involved, an AFLP silver-staining technique system fitting for persimmon genomic DNA analysis was established. Restriction digestion of genomic DNA was performed by using two restriction enzymes, around 450 ng DNA digested with (3 units) of EcoR I and Mse I enzymes respectively in a reaction volume of 20 μL, and incubated at 37℃ for 4 h. Double-stranded adaptors were legated to the restriction fragments at 37℃ for 10 h (or overnight), then transferred to 65℃ for 10 min, after that the digested-legated DNA fragment was diluted (5 folds) with TE buffer and used as templates for pre-amplification. The pre-amplification was carried out using an EcoR I and an Mse I site primer, both without selective bases. The pre-amplification products were diluted (5-bold) with TE buffer and used as starting material for selective amplification. Then selective amplification was carried out using an EcoR I site primer and a Mse I site primer with three selective bases, respectively. At the end of the selective amplification, the samples were denatured by adding 10 μL loading buffer and heated up at 95℃ for 10 min, then cooled on ice immediately. The selective amplified fragments were analyzed by gel electrophoresis. 6.5 μL of each sample was loaded on 6% acrylamide/bisacrylimade gels, using the AFLP silver-staining protocol. The procedure includes discoloring, rinsing, staining, developing, fixing and drying.