| The applicaton of molecular biology in the diagnosis of aquaticanimals is more widely, so the effective preservation of nucleic acid inanimal tissue becomes the key to subsequent molecular biologyresearch..A kind of safe and convenient preservation solution for aquaticanimal will be convenient for the collection of samples and the subsequentmolecular research.In this paper, we choosed Litopenaeus vannamei as the research objectand got several preservation through the combination of differentammonium salt and ethanol. We would like to get a best formula ofpreservation solution for aquatic animals. We put ammonium acetate,ammonium formate and ammonium sulfate respectively in0%,30%ethanol,50%ethanol and70%ethanol until to saturated state. Then weadded EDTA and sodium citrate and adjusted different pH(5.2,6.0,7.0). Atlast we got36kinds of preservation solution to save RNA solution at28℃.The result showed that the saturated ammonium acetate which choosed 70%ethanol as solvent and the saturated ammonium sulfate which choosed50%and70%erhanol as solvent had best RNA preserving effects after3days.We prepared saturated solutions of ammonium sulfate and ammoniumacetate in a serial of solvents with different ethanol concentrations. Theosmosis and RNA preserving effects in shrimp tissue preserved with thesesaturated solutions at normal temperature were tested. The results showedthat ammonium acetate has much more solubility in ethanol solution thanammonium sulfate does. And the molarity of ammonium ion of ammoniumacetate is steadier in0—80%ethanol. Ammonium acetate penetrates intothe tissue of intact shrimp faster and reaches a higher concentration thanammonium sulfate does. RNA with better integrity, higher copies, andlonger half-life period were achieved in the tissue preserved in more than70%ethanol, saturated ammonium sulfate in the solvent of50%and70%ethanol, saturated ammonium acetate in the solvent of50%ethanol.According to abovementioned results, we recommended that the saturatedammonium acetate in50%ethanol as the solvent (pH6.0) can be used assample preserving solution to preserve RNA in shrimp tissue at normaltemperature. It will be a simple and effective method for field sampling andtransportation at normal temperature.In this paper, we choose wood toothpicks and bamboo toothpicks as tools to explore a kind of simple tool to extract nucleic acid rapid. Thetoothpicks were processed by amination and quaternary amination toextract DNA respectively from DNA solution in different concentration andtissue homogenate. In addition, we choosed three kinds of elution buffer torecover DNA. The result showed that TE buffer(pH8.0) preheated at70℃recovered DNA at highest concentration. The bamboo toothpicks withoutchemical modification can extract DNA from DNA solution at lowerconcentration and the recover effect from tissue homogenate was betterthan FTA card which is expensive. So we recommend TE buffer(pH8.0)as the elution buffer and the toothpicks without chemical modificaton as asimple tool to extract DNA from tissue. |
