ࡱ> 463] bjbj * !b!bN8H$l|b($!!!6`XT ^L0|"k~""\!!|"B : Yeast DNA isolation This produces very high quality DNA for southern blots. For PCR templates use the extended yeast colony PCR protocol instead. Harvest 2ml stationary phase yeast culture in a 2ml tube Spin 1 min at 2,500g, re-suspend pellet in 1ml 50mM EDTA Spin 1 min at 2,500g, re-suspend pellet in 250l Buffer A (with DTT and lyticase) by gentle pipetting, incubate at 37( for 45 minutes Spin 5min at 1,000g, re-suspend pellet in 400(l buffer B (including RNase A) by gentle pipetting, incubate 30 min at 37( After this point, handle sample only with cut tips. Do not vortex at any point. Add 4(l 20mg/ml Proteinase K, mix by inverting gently 6-8 times, incubate 30 min at 65( Add 160(l 5M KOAc, invert to mix and incubate 30 minutes on ice Spin 10min at 10,000g at RT, pour the supernatant into a 1.5ml tube Add 500(l pH8 phenol/chloroform, mix on wheel for 30min, spin 10 min at 10,000g, extract with a cut blue tip Add 0.4ml isopropanol, invert to mix Spin 10 min 10,000g at RT, wash pellet with 70% ethanol, dry 10 min at RT Add 20l TE incubate overnight at 4( Next morning flick gently to mix (be gentle with this!) Handle samples with cut tips to maintain DNA integrity, store at 4(. It is almost impossible to accurately pipette or quantify the genomic DNA as it too gloopy, and the solution is never heterogeneous. Instead, for a southern digest 10l DNA per lane, then ethanol precipitate, then quantify. Buffer A: 1.2M sorbitol 50mM EDTA 10mM DTT (added fresh from 1M stock) 340U/ml lyticase (added fresh) Buffer B: 0.3% SDS 50mM EDTA 100(g/ml RNase A (added fresh from 20mg/ml stock) TE: 10mM Tris pH8.0 1mM EDTA RNase A: 20mg/ml in H2O Heat for 15min at 95( to inactivate residual DNase Store at -20( Lyticase: 8.5U/l lyticase 10mM Na Phosphate pH7.0 50% glycerol Weigh out a few mg solid lyticase (Sigma L4025, our low grade material not the super high quality stuff used for PFGE), calculate the required amount of buffer (the activity per mg should be written on the side of the pot) and dissolve by pipetting. This solution is not very stable (it will last a maybe a year at -20(), so dont make too much at once. Store both powder and solution at -20(.      FILENAME Yeast DNA Isolation v3.6 Houseley lab  PAGE 1 ,N      ! ) 0 5 Y f m o { } ~ ÿûǧÜûûûûhi$ jmhAhty jhAhiphNV"h'ChvhJAAh>hs&hO_hwhRO hROhROhGqhkc hZhZhNV"6 hZ6 hN6hZhZ6hA9   Y Z 9 :  0^`0gdO_ 0^`0gd>gdw     X Y Z _ `  : @ A B D H O Y n {     % & h$ZRhkc h jmhJAAhNhX jmhAhJAAhty jhO_ jmhO_hNV"hNV"6 hNV"6hNV"hA jhAh>hO_hi$=  > ? w x $0ef{gdO_gdN& < = L \ _ v x D e { 67Xeiz{>JT`ghڸڸڸڱ h4'ht hw) hNhdZ jhdZ jhN hNH*hBM jmhO_hO_hdZ jhZhZh$ZRhXhN jht ht A)$a$gdU.^gddZȵ h4'ht jhO_0JU*h'C0JmHnHu* hO_0JjhO_0JUh[@hGqhkc h'CmHnHuhO_jhO_Uh:jh:U(/ =!"#$%  s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ OJPJQJ_HmH nH sH tH D`D NormalCJOJQJ_HmH sH tH 44  Heading 1$@&DA`D Default Paragraph FontViV  Table Normal :V 44 la (k (No List 4>@4 Title$a$5CJ(4@4 uHeader  9r 4 @4 uFooter  9r .)@!. u Page NumberH2H +@ Balloon TextCJOJQJ^JaJPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w<    @@MMMP &     +AHJP!8@0(  B S  ?PXdmBJ  o~  33YmXln %%\vVXJT`  YmXln %%\vVXJT`  QPkt ~CGqip>idkc )!NV"a$ao$4'U.L5v=+@q@JAAzO$ZRT 0VdgXZdZ0[>\^O_T,dPxei2uty8NO s&:vvf{W .\'S'Q>fj!ROi$BM.JU i[@u2DXw)AO'Cpw@ffff @UnknownG*Ax Times New Roman5Symbol3. *Cx Arial3*Ax Times5. .[`)TahomaA$BCambria Math"1h{fBeGBeG4LiLi!42qHP?A2!xxkX Yeast DNA isolationDavid Tollervey Michelle KingOh+'0  8 D P \hpxYeast DNA isolationDavid TollerveyNormalMichelle King52Microsoft Office Word@_B@6@*@|OLi՜.+,0 hp  University of Edinburgh Yeast DNA isolation Title  !"$%&'()*,-./0125Root Entry FfT71Table"WordDocument* SummaryInformation(#DocumentSummaryInformation8+CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q