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Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,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,(from 10X Liquid Concentrate 4L, Amresco),(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,Gel runs,- Band cupping & overloading,- Streaking,- Poor separation at high MW,5 ul,1.5 ul,EtBr,leaching,200 bp,300 bp,TBE (new),15 min,600V,Gel runs,- Similar results, somewhat more pronounced 600 V,5 ul,EtBr,leaching,200 bp,300 bp,Sodium-borate,15 min,600V,Gel runs,- Bands are straight,- Minimal streaking,- Good separation at high MW,6 ul,2 ul,6 ul,12 ul,EtBr leaching,Sodium-borate (used 1X),10 min,600V,Gel runs,- Similar results to 600 V for 15 min, except,- Ethidium concentration ok,- High MW bands not as well separated,6 ul,2 ul,EtBr,leaching,100 bp,200 bp,Sodium-borate (used 1X),15 min,500V,Gel runs,- Similar results to 600 V for 10 min, except,- High MW bands are well separated,6 ul,2 ul,EtBr,leaching,100 bp,200 bp,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,15 min,600V,15 min,600V,10 min,600V,15 min,500V,TBE,SB,200 bp,200 bp,200 bp,300 bp,100 bp,100 bp,Bands are weak due to leading edge effect.,Bands are stronger; less time for migration.,Bands are stronger; more time, but lower voltage.,Sodium-borate (new),15 min,600V,5,6,12,ul DNA,ul DNA,TBE (new),45 min,200V,Overloading potential of,TBE,vs. SB gels,TBE (new),45 min,200V,SB (new),15 min,600V,SB (used 1X),10 min,600V,SB (used 1X),15 min,500V,Middle 2 rows of gels,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,Note: EtBr leached into tray buffer from prior 3 gels apparently,“restained” the leading row of samples,100 bp,Excessive Voltage & SB Gels,Sodium-borate (used 1X),10 min, mA increased by Gel melted!,900 initial-V,750V,SB (10 min): EtBr leaching in leading row of each gel,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,Sodium-borate (used 1X),15 min,500V,Gel runs,High-throughput with SB gels,Sodium-borate (new),7 min,600V,Gel runs,Pop Plate #2,Pop Plate #1, Pop Plate #3,300 bp,500 bp,300 bp,500 bp,Sodium-borate (new),7 min,600V,Gel runs,Pop Plate #2,Pop Plate #1,300 bp,500 bp,300 bp,500 bp,Note:,Band resolution would be even,better if run 300-400 V for 15.,Sodium-borate (SB) Limitations,1) No advantages to 1/2X SB,- runs significantly more slowly,- gels dont look as good as with 1X SB,2) Voltage for 25 cm gels:,- 600 V (24 V/cm gel): quality is still good,- EtBr leaches from leading row by 15 min 600V.,- 700 V (28 V/cm gel): quality deteriorates,- 900 V (36 V/cm gel): gels melt,3) Gels 3 kb):,- 5 mM lithium acetate,Sources for SB media,Powder:,Make 40X solutions in glass bottles from either:,A) 5 mM disodium borate decahydrate; or,B) 10 mM sodium hydroxide pHd with borate, as follows:,- 16 g NaOH,- 73.52 g H,3,BO,3,- Stir ,cool, qs to 1000 ml ( pH 8.5 RT).,- Solution must be heated to dissolve the boric acid.,- If precipitates form in 40X solution, simply reheat to dissolve prior to 1X dilution,.,2) 20X SB:,Sodium boric acid conductive medium,from,Faster Better Media,.,- 07Feb08 prices, shipping is extra.,- 1X $1.40/L: $28, 1 liter bottle.,-,1X $1.14/L: $91, 4 liter box, spigot.,-,1X $0.85/L: $170, 10 liter box, spigot.,-,1X $0.72/L: $289, 20 liter box, spigot.,
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