Bio-Rad Trans-Blot® SD Semi-Dry Transfer Cell - Manual (Page 5)

2
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 correspond-
ing percentage agarose gel with 0.5x TBE (i.e., for genomic DNA use 0.7%, plasmid
digests use 0.8% to 1%). We have found that optimal transfer efficiency of plasmid and
genomic DNA is achieved using a 0.7% agarose gel, but higher percentage gels can be
used with a slight decrease in transfer efficiency. To insure proper electrode contact, it is
important to pour a 6 mm thick gel. If using a standard gel tray, such as Bio-Rad's UV
transparent tray, refer to the chart below to determine the proper volume of agarose need-
ed to pour a 6 mm thick gel. If your gel trays have different dimensions, calculate the
volume of agarose required to pour a 6 mm thick gel. Allow the agarose to gel for approx-
imately 30 minutes. Load the gel with your DNA samples and electrophorese in 0.5x
TBE buffer.
Gel Dimensions (cm) Volume Agarose (ml)
7 x 10 42
10 x 15 90
15 x 15 135
15 x 20 180
3. Following electrophoresis, the gel can be stained with ethidium bromide, in 0.5x TBE, and
photographed. The gel should remain in 0.5x TBE before transfer to keep the gel prop-
erly equilibrated for semi-dry transfer.
Section 3
Preparation for RNA blotting
1. For screening 3.5 kb and smaller RNAs, prepare a 1.2% agarose gel with 1x MOPS and
1.8% formaldehyde. See Appendix for formulas.
2. Aqueous samples of total RNA are heated to 65 °C for 10 minutes, then added to load-
ing buffer. Loading buffer consist of 50% formamide, 6.5% formaldehyde, and 1x MOPS.
Typically 5-10 µg of total RNA are loaded per lane. Samples are then heated to 55
°C for
15 minutes; mix 1/10 volume of dye with each sample before loading.
3. Gels are electrophoresed in a submarine apparatus in 1x MOPS buffer until the bro-
mophenol blue dye has migrated ² 10-14 cm. (This usually takes 3-4 hours at 4-5 V/cm .6 )
4. Following electrophoresis, the gel is immersed in at least 5 gel volumes of 0.5x TBE
buffer containing 0.1 µg/ml EtBr for 30 minutes, 7 changed to 0.5x TBE without EtBr for
another 30 minutes. This removes the formaldehyde, stains the gel, and equilibrates the
gel for transfer in one step. The gel may be photographed under UV illumination after
equilibration. The ribosomal bands can be marked at the edge of the gel by stabbing the
gel with an India ink-filled syringe.
The gel should remain in 0.5x TBE prior to transfer to keep the gel properly equilibrat-
ed for semi-dry transfer.