Tail Preparation and PCR Protocol Note: For best results in DNA extraction, tails should be prepped on the

same day as they are clipped. This will yield a lar e amount o! DNA. The tail sample should be approximately " cm or up to the #.$ mar% on the $.& m' centri!u e tubes. ( typically mar% the tubes with both the number o! the animal clipped as well as with an F i! they are F')* animals and a + i! they are +(* animals !or better clarity.

Tail Prep Add &## u' o! the tail lysis bu!!er to each tube. Per!orm a $:, dilution o! the stoc% solution o! -# m .m' Proteinase + to recei/e a & m .m'. Add 0.& u' o! this & m .m' solution to each tube. 1eat o/erni ht with the heater at && de rees C and the roc%er at $& de rees orientation. Tape tubes into the metal holders so they don2t !all out and place the metal holders such that the tubes are on their side. )n the second day, remo/e tubes and /ortex. Centri!u e tubes !or $& minutes at room temperature at $0### rpm. Durin this time, label a second set o! tubes and add &## u' o! (sopropanol to each tube. Remo/e tubes !rom centri!u e machine, and pour supernatant into the newly prepared tubes with isopropanol in them. 3ha%e each tube well and ma%e sure that the DNA cloud is /isible. Centri!u e tubes !or $# minutes at room temperature at $0### rpm. Remo/e tubes !rom centri!u e machine and discard supernatant. Add 0## u' o! 4#5 6T)1 to each tube. Centri!u e tubes !or & minutes at room temperature at $0### rpm. 7sin a Pasteur pipette, brea% o!! the lass tip and place a - u' pipette tip o/er it. 1oo% this up to the /acuum in the !ume hood. Remo/e tubes !rom centri!u e machine and aspirate to remo/e all 6T)1 !orm the tube, ta%in care as to not /acuum the pellet into the pipette. 'et dry !or approximately $# minutes 8the DNA will be a white color when wet and will become pro ressi/ely more clear at it dries !rom the outside in9. :hen dry, add -## u' dd1-) to each tube and place tubes in heater at && de rees Celsius. 1eat until the pellet is resuspended into the solution. 8Typically ( per!orm the second day DNA extraction in the mornin and then in the late a!ternoon ( do the PCR o! these samples to run o/erni ht9. :hen resuspended, remo/e tubes !rom heater and /ortex. Per!orm a $:-& dilution in !resh tubes 8( typically do , u' DNA and ;< u' o! dd1-)9.

PCR Prep each PCR tube with the !ollowin : $ u' DNA !rom $:-& dilution prepared " u' F')*.+(* !orward primer " u' F')*.+(* re/erse primer , u' =oTa> solution , u' dd1-) Typically ( ma%e a master mix o! the last , and add ; u' to each tube that has $ u' DNA inside. ?ix thorou hly. Place in PCR machine. For F')* animals, best results occur when runnin the pro ram labeled as +(*. For +(* animals, best results occur when runnin the pro ram labeled as T)7C1D):N. =el Prep Add 4.& A arose per &## m' o! $ x T@6 in a $ ' bea%er. &## m' !ills the lar er el box. For the smaller el box, use approximately $&# m' 8-.-& A arose9. 1eat solution in microwa/e !or & minutes 8- " minutes !or smaller el9. 'et el set !or about ,& minutes. Remo/e combs and !ill el box with $ x T@6 bu!!er solution so that el is completely co/ered. Add 0 u' o! DNA loadin el to each PCR tube. ?ix thorou hly with pipette and load slowly into el wells. Run el at ;; A, -## amps 8minutes doesn2t matter9. ( typically run the lar e el !or 0 " to , hours. The smaller el box !inishes in $ " hours. :hen !inished, ta%e el out and place in container. Cut el as desired to !it into containers. Prepare a mixture o! &# m' Tris solution at p1 4.&, $# m' & m? 6DTA solution, and & u' 3B@R DNA el stain. For the lar er container, use C# m' Tris solution at p1 4.&, $< m' & u? 6DTA solution, and C u' 3B@R DNA el stain. Place in !oil 8DNA stain is li ht sensiti/e9 and place on roc%er at $#D$& de rees orientation !or C#D;# minutes. Ta%e el to typhoon scanner 8in what used to be the library behind the @onney con!erence room9. Place el in scanner and scan accordin to the letters and numbers it ta%es up. 7nder settin s, chan e to !luorescence. )pen the 3etup menu. ?a%e sure it is set to: &-#, P?T is <##, @luee ,CC and the >uality is medium. Clic% )+. Further down !rom where you chan ed to !luorescence, be sure the orientation matches how you placed it into the machine, the sample is N)T set to be pressed. -## microns is

!ine 8the lower the microns the more !ine >uality you et in the scan but the lon er it ta%es to scan9 and you chan e it !rom platen to E0 mm. 3can el and open the el !ile. @ands can be dar%ened !or better /isibility. Print el and mar% accordin ly. For both F')* and +(*, the hi her o! the two bands is the mutated allele, the lower is !or the wild type. +(* and F')* animals ha/e the hi h band only, but will typically ha/e a tail that will ma%e the band much thic%er, but will not o as !ar down the el as the wild type band.