Rapid alkaline blot-transfer of viral dsRNAs |
| |
Authors: | J K Li B Parker T Kowalik |
| |
Affiliation: | 1. Biostabilization Laboratory - Lower Saxony Centre for Biomedical Engineering, Implant Research and Development, Stadtfelddamm 34, 30625 Hannover, Germany;2. Unit for Reproductive Medicine - Clinic for Horses, University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany |
| |
Abstract: | The double-stranded genomic RNAs of reovirus and bluetongue virus can be transferred very efficiently from either sodium dodecyl sulfate-polyacrylamide gels or NuSieve agarose gels onto several nylon membranes. After a brief acid depurination treatment, viral dsRNAs from the gels are transferred at room temperature using 0.2 N NaOH as the transfer medium. Four blots can be obtained within 1 h and each blot contains 15-20% of the input RNA sample. These blots can be used immediately without baking in vacuo. Less than 5% of the "fixed" dsRNAs are removed after repeated washings of the membrane blots. As little as 10 pg of the genomic dsRNA segment can be detected in this alkaline Northern blot. A 20- to 50-fold increase in resolution and sensitivity over traditional Northern blots is routinely achieved. These alkaline blots can be reused 6-10 times after appropriate strip washing and proper handling. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|