Southern hybridization of RT-PCR clone
(antibody light chain) OBJECTIVE OF SOUTHERN BLOTTING: To confirm the RT-PCR clone (white colony growing on kanamycin) contains the antibody light chain gene. 13A CONCEPT OUTLINE We will probe plasmid DNA, so not much DNA is required; it will be digested with only one restriction enzyme (EcoRI). When you do a Southern of a higher organism's genomic DNA, at least 10 g is cut, often with several restriction enzymes in parallel (typically 6cutters to generate large DNA fragments). On an agarose gel, digested eukaryotic genomic DNA will produce a smear because of the thousands of bands of various sizes which are generated. The Southern will be able to detect a single small fragment of DNA in the smear. In our experiment, the pattern will be infinitely simpler.
START: Miniprep plasmid DNA using Qiagen miniprep kit protocol ↓ Digest with EcoRI (12.5 uL DNA; 1.5uL buffer; 1uL EcoRI; 30-60 minutes) 13B BASIC EXPERIMENTAL OUTLINE I. For a Southern blot, tight, well-resolved bands are necessary for a clean result. Therefore gels are typically run at a low voltage for a long time. Before transfer to the membrane, DNA in the gel must be denatured using NaOH. ssDNA is required to allow probe hybridization. WEAR GLOVES to prevent oils from your hands getting on the membrane or gel RUN GEL (With ladder, positive and negative controls) Pour 1.6% agarose gel ↓ Aliquot ladders and control plasmid ↓ Add 1 l loading dye to all samples (controls and digest) ↓ Load 10 l from each tube ↓ Electrophorese at 60 volts for approximately 2 hours II. DENATURE AND NEUTRALIZE DNA Transfer gel to container with denaturing buffer ↓ Agitate for 15 minutes ↓ Pour off denaturing buffer and repeat 1
↓ Pour off denaturing buffer and add distilled water to rinse briefly ↓ Pour off water and add neutralizing buffer ↓ Agitate for 15 minutes ↓ Pour off neutralizing buffer and repeat III. SET UP BLOTTING APPARATUS AND TRANSFER DNA TO NYLON MEMBRANE {see diagram at end of handout} DO NOT touch membrane with bare hands! Obtain membrane, label it (ballpoint pen only) with name & mark a corner for orientation; wet it by briefly floating in distilled water ↓ Put glass plate on rubber stoppers in bottom of blotting container; add 10X SSC buffer to fill ↓ Place two filter papers together and soak in SSC, then place on tray top. Ends of this paper MUST be submerged in SSC buffer as this is the "wick" which pulls buffer up into the gel. ↓ Remove gel from neutralizing buffer and place face down on the wet filter papers ↓ Place membrane on top of gel, align carefully and rub out air bubbles ↓ Put 4 strips Parafilm sheet wax around membrane edge to prevent short-circuit ↓ Center two pieces of filter paper on top of membrane ↓ Center a stack of paper towels on top of filter paper ↓ Add weight (flask full of water) and leave overnight

下一页