Pulse Field Electrophoresis

Pulsed field electrophoresis :-separation of large UNA molecules by applying to ageb Matrix an electric fuld that periodically changesdirection.1982, Schuoutz introduced the concept that DNA moleculeslarger than 50kbcan be separated by using 2 alturatingelectricfieldsperiodic changing of field direction, the various lengthsof DNA react to the charge at differing ratesThat islarger pieces of DNA will be slower to realigntheircharge when field diruction is changed, whitesmallerpieces will be quicker. over the couse of timewiththe consistent charging of eludions , each will begin to separate move of more even at very large qu lengthsThus large DNA a separatedInstead of constantly running the voltage in one direction,the vollage is priodically switched among three directionsone that runs through the central axis of the gel and twothat run at an angle of 60 degrees either sideidentify b-add to buffer - make suspensionadd molter agaiose - to make pluy.Lyse cellsEnzyme digestElectrophonesisDensitometrySpecial Spectiophotometu that measures light transmittedthrough a Solid Sample Such as cleand / transparent butStaned gel. using the optical density measuremate,the densitometer represents the bards a peak. 2nd electionicS2 sigletstateJablonskisame ofinterestrongT, ( excuted triput state)phosphorescencefurrescenceSodg& 23InGroundsinglet stateGround state minimum valuewhen extunal raciation / EMI fallson the molecule,absorbsenergy Elationic transition takes I place Molecules transitto a higher energy state Remains for a certain time.Returns back of to ground State by relearing energyemitted wave length is greater than absorbeda21<23Sometimes it goes to T1Thenfall backtogroundstatephosphoresence.Flue- immidiate emission of absorbed radiationsPhosphu - delayedanaMTransmitted beanangle a kept at are 90"fluosent reading90unaftered lightCuvetteemittedlitterbeamDelectlightsource islandTR = 18.24N,H=?w1/2 = 0.58L = 30Problemsto HETP = L/NN= 5.55 ( tR 72w/1218.242= S.SS X0.58N = 5476.H = IN= 3015476= 0.005cm 10.05mmR=tR2 tRI CR I greater than 2)1/2 (witw2) (2 = N 2000N= 16 l tR )2wbHigh log P. , high lipophilicilynonpolarRp SP non polar I Nb polarEluting laster - polarslower - nonpolar o'TMSCH3 - CTO -CH3243&102 peak 2L CH3) same en. C=O -different ersBenzene , autoneSingleautaldehyde - 2CH3 - E-HTMSstd Y ITMSSince Silicone less eletx electionegative than caubonH2 of TMS are highly election density em-highly shilled with the signals of other compoundslow resonance frequency and rarely criterfere12 equivalent H2 Shocus as a single peaksignal as strong.eary toquite crust and remaved with bp of 27°.1IN III CCH2OCH343Methyl aalateCM3 isCH3simethys butaneC=CM3as21