Sodium-borate(SB)vs.TBEforgelelectrophoresisByusing.ppt

Sodium-borate (SB) vs. TBE for gel electrophoresis By using SB for both gel and running buffer, we can: 1) Run gels faster at higher-throughputs 2) Get better quality gels -- Sharper bands -- Fully separated bands in 100-bp DNA ladder -- Better sample band separation size estimates -- Harder to overload with DNA 3) Save $ -- TBE = $0.54/L (from 10X Liquid Concentrate 4L, Amresco) -- SB = $0.11/L (from disodium borate decahydrate powder, 500 g, Fisher);Overview of Talk – 30 slides PCR product: 543 bp Alu (Owl monkey). -- Compare TBE SB at various voltages running times -- show whole gels; -- compare effects of EtBr leaching -- DNA overloading potential of TBE vs. SB -- focus on middle 2 rows in gels; -- Remelting of SB gels -- SB gels at excessive voltage -- Recap TBE vs. best SB parameters -- High-througput gels -- Summary slides;TBE (new) 45 min 200V;TBE (new) 15 min 600V;Sodium-borate 15 min 600V;Sodium-borate (used 1X) 10 min 600V;Sodium-borate (used 1X) 15 min 500V;Ethidium Bromide Leaching 1) Primarily affects leading row of DNA samples 2) To Reduce effect: a) move gel section away from edge of UV illuminator b) crop photo, click on “transform”, and adjust “gamma” slide ;45 min 200V;;TBE (new) 45 min 200V;Remelting of SB gels ;Sodium-borate (remelted 3 times) 15 min 600V;Sodium-borate (remelted 3 times) 15 min 600V;Sodium-borate (remelted 3 times) 15 min 600V;Excessive Voltage SB Gels ;Sodium-borate (used 1X) 10 min – mA increased by 100 600V – Temperature increased 3oC;750 V (10 min);900 initial-V;Recap -- standard TBE vs. best SB parameters Note: Remember to make BOTH the gel and the running buffer from 1X SB media. ;TBE (new) 45 min 200V;Sodium-borate (used 1X) 15 min 500V;High-throughput with SB gels;Sodium-borate (new) 7 min 600V;Sodium-borate (new) 7 min 600V;Sodium-borate (SB) Limitations 1) No advantages to 1/2X SB -- runs significantly more slowly -- gels don’t look as good as with