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All in One Restriction Enzyme Digest and Loading Buffer
From Sci-Mate Wiki
Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Analysis (Electrophoresis, Southern Blotting)| Editor: Andrew Lew Branch: Protocol-Wiki Type: Solution Recipe Intention: For Publication Read/Write Permissions: Open Access | Authors : 16.03.2009 - Andrew Lew |
This combination of 10X universal digest buffer plus loading dye can be used as a single buffer for both restriction enzyme digestion and then immediate loading of samples onto agarose gel. As a universal digest buffer it saves the time and effort of separate digests by allowing efficient DNA cutting with a wide variety of restriction endonucleases in a single buffer. More time is then saved due to the presence of loading dye in the buffer, allowing samples to be loaded directly into a gel following digestion without the need to subsequently add loading buffer. Repeated analysis and experience has shown no significant difference in digestion or subsequent cloning efficiency.
[edit] FOPA 10x Solution Buffer Contents
| | FOPA 10x | Final Molarity |
| Ficoll 400 | 2 g | 0.5 mM |
| Potassium acetate | 0.49 g | 500 mM |
| Magnesium acetate | 0.21 g | 100 mM |
| 1M tris acetate pH7.5 | 1 ml | 100 mM |
| Orange G | 15 mg | |
| Distilled Water | qv to 10 ml | |
[edit] References
This method was first described in: Corbett AJ, Brady JL, Lew AM. 2002 A single buffer that universally serves both restriction digestion and loading. Mol Biotechnol. 21:51-5.
