Trans-Blot® SD DNA/RNA Blotting Kit Instruction Manual Catalog Number 170-3957 For use with the Trans-Blot SD sem-dry electrophoretic transfer cell (catalog numbers 170-3940, 170-3948, 170-3949) For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Table of Contents Page Section 1 Introduction .................................................................................................. 1 1.1 Specifications.............................................................................................................. 1 Section 2 Preparation for DNA Blotting .................................................................... 2 Section 3 Preparation for RNA Blotting ....................................................................
Note To insure best results from the Trans-Blot SD DNA/RNA Blotting Kit, become fully acquainted with its instructions before using the kit. It is also recommended that you follow the protocol as closely as possible in order to achieve optimum results. Bio-Rad recommends that the kit component (gel support frame) be cleaned with a mild, laboratory cleaner (such as Bio-Rad Cleaning Concentrate, catalog number 161-0722) and rinsed thoroughly with distilled water, before use. Catalog No.
Section 1 Introduction The standard capillary transfer of DNA/RNA from agarose gels to blotting membranes is usually an overnight procedure.1-3 The Trans-Blot SD DNA/RNA blotting kit, used with the Trans-Blot SD semi-dry transfer cell, allows blotting of DNA and RNA from agarose gels to nylon membrane in only minutes, without any gel pretreatments. The Trans-Blot SD semi-dry electrophoretic transfer cell generates a high field strength (V/cm2) which facilitates rapid transfers.
Section 2 Preparation for DNA Blotting 1. Prepare enough 0.5x TBE transfer buffer for use in both the horizontal agarose gel electrophoresis and the semi-dry transfer (depending on size of gel). See Appendix for directions. 2. Depending on the size of the DNA fragments to be electrophoresed, pour a corresponding percentage agarose gel with 0.5x TBE (i.e., for genomic DNA use 0.7%, plasmid digests use 0.8% to 1%).
Section 4 General Assembly of Unit for Transfer 1. Saturate two pieces of precut Extra Thick Blot Paper (provided with the kit) and precut Zeta-Probe® GT membrane (also provided with the kit) or any other transfer membrane in 0.5x TBE buffer. Equilibrate the transfer membrane for at least 10 minutes. The transfer membrane and blot paper must have the same dimensions as the gel for proper transfer to occur.
. Place the safety cover onto the unit and plug the Trans-Blot SD cell into the power supply. Be sure to maintain normal polarity of the electrodes, i.e. red lead to red outlet and black lead to black outlet. Caution: Do not reverse the electrode polarity. This will damage the stainless steel cathode. 8. Turn on the power supply. The power conditions and transfer times will vary depending on what type and size of DNA/RNA you transfer.
b. Power conditions during transfer may have changed. It is important to have constant current during the course of the run. If the buffer is less concentrated than 0.5x, higher voltage will be required to maintain the recommended current. If the voltage limit is not set high enough, the current will drop below the optimum range during the run, thereby reducing DNA/RNA migration. Readjust the power supply parameters. c.
Section 6 Appendix 10x TBE (per liter*), pH 8.3 Quantity Final 0.5x Concentration 108 g Tris base 55 g boric acid 40 ml 0.5 M EDTA, pH 8.0 44.5 mM Tris base 44.5 mM boric acid 1 mM EDTA To make a 0.5x TBE working buffer, mix 50 ml of the 10x TBE stock per 1 liter of deionized, distilled water. * One liter of Bio-Rad premixed 10x TBE is included with the kit. 1x MOPS, 1 L Quantity Final 1x Concentration 4.186 g MOPS 0.41 g sodium acetate 2 µl of 0.5 M EDTA, pH 8.
Section 7 Equipment and Accessories Kit Components 161-0733 Tris/Boric Acid/EDTA, 1 L 170-3960 Extra Thick Blot Paper, 15 x 20 cm, 30 Gel Support Frame Trans-Blot SD DNA/RNA Blotting Protocol DNA/RNA Blotting Kit Accessories 170-3958 Extra Thick Blot Paper, 10 x 15 cm, 30 170-3959 Extra Thick Blot Paper, 15 x 15 cm, 30 170-3960 Extra Thick Blot Paper, 15 x 20 cm, 30 Trans-Blot SD Electrophoretic Transfer Cells 170-3940 Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell, complete unit 170-3948
Section 8 References 1. 2. 3. 4. 5. 6. 7. 8. 9. Southern, E. M., Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98, 503-517 (1975). Alwine, J. C., Kemp, D. J., Parker, B. A., Reiser, J., Renart, J., Stark, G. R. and Wahl, G. W., Methods Enzymol., 68, 220 (1979). Seed, B., Nuc. Acids Res., 10, 1799 (1982). Lane, K., Zoia, O. and Davidson, J. M., BioTechniques, in press (1991). Zeta-Probe GT Instruction Manual, Bio-Rad Laboratories (1990).
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