6.2 DNA Blotting
(For acrylamide gels with DNA 250 bp to ~1 kb)
Electrophoresis Run on a Polyacrylamide Gel
1. Prepare the stock electrophoresis 5x TBE buffer (Section 5). Dilute the stock to 1x.
2. Mix 10–15 µl of the sample with 5 µl of 5x dye buffer, heat to 65 °C for 5 min and load
on a gel.
3. A 5% PAGE gel can separate DNAs from about 250 to 1,000 bp.
4. Run the gel in 1x TBE buffer at 100 V for 1–2 hours.
Standard Blot to Zeta-Probe
1. From the 5x TBE electrophoretic buffer, dilute the stock to 0.5x (Section 5) and pre-chill
1 L of the buffer.
2. Equilibrate the gel, extra thick blot paper, and Zeta-Probe membrane in 0.5x TBE buffer
for at least 15 minutes.
Note: Zeta-Probe membrane will bind non-denatured nucleic acids. Therefore, denaturing
is not mandatory before transferring. If non-denatured nucleic acids are transferred, the
blotted Zeta-Probe membrane must be treated with NaOH prior to hybridization. Refer to
the Zeta-Probe membrane instruction manual.
3. Assemble the sandwich as described in Section 4.2.
4. Run the transfer at 400 mA for 1 hour (voltage should not exceed 25 volts).
5. After transfer, separate the membrane from the gel, and rinse the membrane briefly in
0.5x TBE buffer.
6. Fix the DNA to the membrane by placing the membrane on several pieces of blot paper
saturated with 0.4 N NaOH for 10 minutes.
7. Rinse the membrane in 2 x SSC for 10 minutes and bake at 80 °C for 1 hour (this is
optional if probing immediately). The membrane is now ready for hybridization. Refer to
the hybridization procedure in the Zeta-Probe blotting membrane instruction manual.
6.3 DNA & RNA Blotting
(For agarose gels with DNA up to 23 kb, RNA up to 3.5 kb)
Refer to the Trans-Blot SD DNA blotting kit instruction manual for transfer protocol and
conditions. DNA or RNA cannot be blotted from agarose gels without the use of the Trans-
Blot SD DNA blotting kit.
Section 7
Properties of Protein Blotting Media
PVDF membrane is suitable for presenting transferred proteins for immuno detection
(Immun-Blot PVDF) or analysis by Edman. It is resistant to tearing and chemicals. Immun-
Blot PVDF is optimized for immunodevelopment with high protein binding capacity (160
µg/cm
2
), but low nonspecific protein binding. This membrane material will resist tearing even
when used in repeated stripping and reprobing applications. Sequi-blot PVDF has the
highest protein binding capacity (170–200 µg/cm
2
) and gives outstanding performance in
protein sequencing applications.
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