From jwbrown@crab Wed Jan 15 02:45:08 1992 Subject: End-labeling DNA fragments End-labeled DNA fragments are used in a wide variety of molecular biology experiments. Doubley end-labeled fragments are useful as MW standards on Southern blots, as probes for filter-binding or gel retardation experiments, or for visualizing small amounts of restricted DNA. Singley end-labeled DNA is used for Maxam & Gilbert sequencing, for S1 analysis, footprinting, primer extension, etc. This method can also be used for 5' end-labeling RNA molecules. 10X Kinase buffer Prep Gel ------------------ ---------------- 0.5M Tris,pH9.5 48.3ml ddH2O 0.1M MgCl2 25ml glycerol 50mM DTT 60mg APS 50% glycerol 16.6ml 29%:1% acrylamide:bis 10ml 10X TBE Denaturation buffer -------------------- 1M Tris, pH9.5 Elution buffer 10mM Spermidine ---------------- 1mM EDTA 0.5M NH4OAc 10mM Mg(OAc)2 Phenol:chloroform 1mM EDTA ------------------- 0.1% SDS 48% chloroform 50% TE sat'd phenol 2% isoamyl alcohol 1 - Digest 10ug DNA with the appropriate restriction enzme (the one you want to label at) overnight in a 30ul reaction. If you desire or need to, a larger reaction can be used, then the DNA should be EtOH precipitated and resuspended in 30ul 1X restriction buffer. 2 - Add 3.5ul 1M Tris, pH8, and 2ul 4u/ul CIAP (calf intestine alkaline phosphatase). Incubate 30 min at 56C. 3 - Add 56ul 2M NH4OAc and 156ul ddH2O. Add 200ul phenol:chloroform, vortex VIGOROUSLY, then spin 1 min. Collect the upper (aqueous) phase and again add 200ul phenol:chloroform, vortex, and spin as before. Collect the upper phase, and add 600ul EtOH and freeze for 5 min in a dry-ice:EtOH bath. Spin for 5 min, discard the supernatant, and add 750ul ice-cold 70% EtOH. Again freeze for 5 min in a dry-ice:EtOH bath, spin for 5 min, and discard the supernatant. Dry the pellet in a rotovac. 4 - Dissolve the pellet (likely invisible) in 20ul ddH2O, then add 20ul 2X denaturation buffer. Incubate for 15 min at 65C (95C for 3' overhangs), and quick-chill on ice. 5 - Add : 6ul 10X kinase buffer, 100uCi gamma-32P-ATP, ddH2O to 59ul, and 20u polynucleotide kinase. Incubate for 1hr at 37C. 6 - Add 56ul 2M NH4OAc and 134ul ddH2O. Phenol:chloroform extract, EtOH precipitate, wash, and dry as in step 3. --- for singly end-labeled fragments --- 7 - Dissolve the dry DNA in 40ul ddH2O, ad 5ul 10X restriction enzyme buffer,and 5ul of the appropriate restriction enzyme. Incubate at the appropriate temperature for 3-5hr. 8 - Add 10ul 10X tracking dye and load onto a 2mm thick 5% prep gel. Electrophorese overnight at 160-180V (for our standard 23cm length gels) in TBE. 9 - Remove one glass plate and cover the gel with saran-wrap. In the dark room, cover the gel with a small piece of X-ray film for 2 min. Develop the film. When the film is dry, cut out the exposed area for the bands you want to elute, then place the film over the gel PRECISELY as it was exposed (use the tracking dye & wells as guides) and mark the saran-wrap with a sharpy where you've cut out the image of the bands. Remove the film, then cut the DNA containing regions from the gel with a razor blade. Dice the gel slice with the razor blade. 10 - Flame-seal a blue 1ml pipette tip & plug the tip with a small amount of glass wool. Put the diced acrylamide in the tip, & wash it to the bottom with 500ul elution buffer. Cover the top with parafilm, vortex, and incubate overnight at 37-42C. 11 - Carefully puncture the sealed-up tip with a red-hot needle, then put the tip point-down into a 4ml snap-cap tube with the top half of the lid in place (cut off the bottom of the lid with a razor blade, leaving a ring that snaps onto the top of the tube). Puncture the parafilm with a red-hot needle, and spin for 1 min in a clinical centrifuge. Add 100ul elution buffer to the tip and spin again. Collectthe eluted DNA from the snap-cap tube and transfer to and eppendorf tube. 12 - Add 1ml EtOH and freeze for 5 min in a dry-ice:EtOH bath, spin for 5 min, and discard the supernatant. Wash 3 times by adding 750ul ice- cold 70% EtOH, freezing, and spinning as before. Dry the final pellet in a roto-vac, and count in a scintillation counter. Elio Vanin, OSU Dept. Biochemistry Maxam and Gilbert, Meth. Enzymol.