sodiumborate (sb) vs tbefor gel electrophoresisby using ：硼酸钠（sb）vs tbefor electrophoresisby使用凝胶.ppt

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1、Sodium-borate(SB)vs.TBEfor gel electrophoresisBy using SB for both gel and running buffer,we can:1)Run gels faster&at higher-throughputs2)Get better quality gels -Sharper bands -Fully separated bands in 100-bp DNA ladder -Better sample band separation&size estimates -Harder to overload with DNA3)Sav

2、e$-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

3、-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 slidesTBE(new)45 min200VGel runs-Band cupping&overloading-Streaking-Poor separation at high MW5 ul1.5 ulEtBrleaching200 bp300 bpTBE(new)15 min600VGel runs-Si

4、milar results,somewhat more pronounced 600 V5 ulEtBrleaching200 bp300 bpSodium-borate15 min600VGel runs-Bands are straight-Minimal streaking-Good separation at high MW6 ul2 ul6 ul12 ulEtBr leachingSodium-borate(used 1X)10 min600VGel runs-Similar results to 600 V for 15 min,except-Ethidium concentrat

5、ion ok-High MW bands not as well separated6 ul2 ulEtBrleaching100 bp200 bpSodium-borate(used 1X)15 min500VGel runs-Similar results to 600 V for 10 min,except-High MW bands are well separated6 ul2 ulEtBrleaching100 bp200 bpEthidium Bromide Leaching1)Primarily affects leading row of DNA samples2)To Re

6、duce effect:a)move gel section away from edge of UV illuminatorb)crop photo,click on“transform”,and adjust“gamma”slide45 min200V15 min600V15 min600V10 min600V15 min500VTBESB200 bp200 bp200 bp300 bp100 bp100 bpBands are weak due to leading edge effect.Bands are stronger;less time for migration.Bands

7、are stronger;more time,but lower voltage.Sodium-borate(new)15 min 600V56 12ul DNAul DNATBE(new)45 min200VOverloading potential of TBE vs.SB gelsTBE(new)45 min200VSB(new)15 min600VSB(used 1X)10 min600VSB(used 1X)15 min500VMiddle 2 rows of gelsRemelting of SB gelsSodium-borate(remelted 3 times)15 min6

8、00VSodium-borate(remelted 3 times)15 min600VSodium-borate(remelted 3 times)15 min600VNote:EtBr leached into tray buffer from prior 3 gels apparently “restained”the leading row of samples100 bpExcessive Voltage&SB GelsSodium-borate(used 1X)10 min mA increased by Gel melted!900 initial-V750VSB(10 min)

9、:EtBr leaching in leading row of each gelRecap-standard TBE vs.best SB parameters Note:Remember to make BOTH the geland the running buffer from1X SB media.TBE(new)45 min200VSodium-borate(used 1X)15 min500VSodium-borate(used 1X)15 min500VGel runsHigh-throughput with SB gelsSodium-borate(new)7 min600V

10、Gel runsPop Plate#2Pop Plate#1 Pop Plate#3300 bp500 bp300 bp500 bpSodium-borate(new)7 min600VGel runsPop Plate#2Pop Plate#1300 bp500 bp300 bp500 bpNote:Band resolution would be evenbetter if run 300-400 V for 15.Sodium-borate(SB)Limitations1)No advantages to 1/2X SB-runs significantly more slowly-ge

11、ls dont look as good as with 1X SB2)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 melt3)Gels 3 kb):-5 mM lithium acetateSources for SB media1)Powder:Make 40X solutions in

12、 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 H3BO3-Stir&heat in 800 ml Nanopure water;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.