ࡱ> 352] bjbj *!b!b1^8H$lb($!]]]]]]]&]20b]8"8"]8"]p]b8"B : Yeast DNA isolation low cell number If cells are fixed in 70% ethanol then wash with 100l 50mM EDTA, otherwise do not wash. To pellet low cell numbers in 1.5ml tubes, add 0.01% Triton. Re-suspend pellet in 50l Buffer A (with DTT and lyticase) by gentle pippetting, incubate at 37( for 45 minutes Spin 5min at 1,000g, re-suspend pellet in 80(l buffer B (no RNase A!!) containing 1l 20mg/ml proteinase K by gentle pipetting, incubate 30 min at 65( After this point, handle sample only with cut tips. Do not vortex at any point. Add 32(l 5M KOAc, flick to mix and incubate 30 minutes on ice Spin 10min at top speed at RT, pipette the supernatant into a 1.5ml tube using a cut tip Add 100(l pH8 phenol/chloroform, mix on wheel for 30min, spin 10 min at 10,000g in hood, extract upper phase with a cut tip Add 250l ethanol and 1l of glycogen, flick to mix, incubate >1 hour at -30 Spin 15min top speed at 4(, wash pellet with 70% ethanol, dry 10 min at RT Add 17l TE incubate overnight at 4( Next morning flick gently to mix (be gentle with this!) For southern blot analysis: Add 2l 10x buffer and 1l restriction enzyme, mix and incubate for ~3 hours at 37( Quantify 2x 0.2l of DNA from the digests using PicoGreen, and put the required amount of digest in a new tube. Add 4l 6x blue loading dye and load onto gel. 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 TE: 10mM Tris pH8.0 1mM EDTA 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.      FILENAME Yeast DNA Isolation low cell number v1.3 Houseley lab  PAGE 2 #%     $ 3 4 7 ; > A D E F N U Y Z [ c d f g i j q s t       # ԺԶбܺhNV"hNV"6 hNV"6hi$ jmhAhty jhAhiphNV"h>hO_h/$hwhvhJAAhROhXhHh@ hAB$%- .   U V - . | } ' ( D E gdN 0^`0gd>gdw# $ & . 5 C D E U V d m s |    , - . 2 5 7 8 ; ? S Z a z { }  & ܪܪ jht ht jh?(h?(hQYOJQJhQYh$ZRhXhhi$ jmhJAAhNh/$h@ hAhO_hJAAhHhXh>@& ' ; < ? @ A K X Y e o | IU./0124578:;=>HIlmnrs{ûٛhUh*mHnHu hXhO_jhO_UhU\jhU\U h4'ht hw) hNhdZhBMhO_hdZhNhXht hX jhF`hF`hH9E ; < = > ? @ Y e %4/0$a$gdU.^gd?(gdO_gdN0134679:<=$a$gdU. h4'ht hU\hO_jhO_0JU*hX0JmHnHu* hO_0JjhO_0JU(/ =!"#$%  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+$G]7O٭Vc:E3v@P~Ds |w< * PP]]]`# &  E 0 ;QXZ`!8@0(  B S  ? #' MU134679:<=#%).dmv|))278;?SUZa{&:<<==..01144=#%).dmv|))278;?SUZa{&:<<==..01144=ns{VUF`kt ~Cip>id@ XNV"a$ao$4'U.6#1K4L5.{9v=+@q@JAAzO$ZR 0VdgXQYZdZ0[>\O_T,dPxeityr8NO s&vvUf{W .\'-S'Q>fj*H!ROi$BMU\U iuDXw)AO/$?(xMpw13@P@UnknownG*Ax Times New Roman5Symbol3. *Cx Arial7.@Calibri3*Ax Times5..[`)TahomaA$BCambria Math"1h1gG1 ,  4..2qHP?A2!xx} Yeast DNA isolationDavid Tollervey Jon HouseleyOh+'0  8 D P \hpxYeast DNA isolationDavid TollerveyNormalJon Houseley10Microsoft Office Word@%@>7@y6@e՜.+,0 hp  University of Edinburgh . Yeast DNA isolation Title  !#$%&'()+,-./014Root Entry F@61Table8"WordDocument*SummaryInformation("DocumentSummaryInformation8*CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q