EMSA_VS_DNA标记.pptVIP

DIG-EMSA ELECTROPHORESIS During probe binding, prepare a 6% non-denaturing TE-polyacrylamide gel as follows (for 10mL gel): Pre-chill 0.5xTE buffer gel to 4?C, then run at 120V prior to loading to equilibrate. Add 1.0uL of 10x DNA loading buffer (orange dye only!!) to binding rxn after 30 min incubation gently mix. Load all rxn to gel run at 120V in the cold room at 4?C to keep from overheating the gel. Stop electrophoresis when orange dye reaches end of gel (or just run it off; orange dye runs at ~50bp of DNA). DIG-EMSA TRANSFER Cut 4 Whatman blotting papers a nylon membrane (Amersham Hy-bond XL) to appropriate size of gel pre-soak all but one Whatman in 0.5x TE buffer. Carefully disassemble the gel plates so that the gel adheres to one of the two plates, and place the dry Whatman paper over the gel. Carefully remove the gel/Whatman layer (the gel should adhere to the paper come off the plate relatively easily) place in 0.5x TE buffer. Place the nylon membrane over the opposite side of the gel, and sandwich the Whatman papers, gel membrane on a semi-dry transfer apparatus; ensure liberal soaking of the entire sandwich with 0.5x TE buffer. ORDER (top-to-bottom): 2 Whatman gel membrane two Whatman bottom half of the transfer apparatus. Roll out any bubbles in the sandwich with a clean disposable pipette. Transfer for 30 min at 300mA of constant current. DIG-EMSA DETECTION Disassemble the transfer apparatus immobilize bound probes by baking for 30 min at 85?C in a dry oven OR by UV crosslinking for 3 min (in Koenig Stratalinker). After immobilization, wash the membrane 2 x 5min in PBST (PBS + 0.1% Triton-X). Block membrane 30 minutes at room temperature in 2% dry milk in PBST. Hybridize anti-DIG-AP Fab fragments (Roche #1093274) to membrane for 30 minutes at room temperature. Wash membrane 3x10 minutes in PBST. Incubate membrane 2 minutes in TMNT buffer (0.1M Tris-buffer [pH 9.5], 0.1M NaCl, 0.05M MgCl2, 0.1% Tween-20). Mix 200uL of NBT/BCIP stock so