ࡱ> >@=a  bjbj .AbAbtS44444HHH8H($Fd=4=44R___F44____'ߕ9!F_h0_7_4__==|B : Yeast spotting assay The day before, leave the plates you will use on the bench overnight (budget 2 plates for each condition) This assumes starting with saturated cultures, but it can be done (and probably better) from log phase cells. For each strain to be tested, label 4x 1.5ml tubes each with 90 l water Measure the OD of your culture Calculate the volume required for 1x106 cells Put this volume of culture in a 1.5ml tube, dilute to 100l final volume From this initial dilution, make 4 serial dilutions of 10 l into 90 l water When making these dilution series, be sure to vortex each tube thoroughly before pipetting For each starting culture you should now have 5 tubes containing 1x106, 1x105, 1x104, 1x103 and 1x102 cells in 100 l Place your first two plates on a spotting template Spot 8 l from each of your 1x106 cell tubes in the left column of each plate Remember to vortex before taking each spot Then spot the 1x105 cell tubes on the next column, and so on Put the lid on the plate but leave on the bench for ~1 hour to make sure the liquid is fully absorbed before putting upside down in incubator Continue with other plates. It is wise to do 2 replicates of each plate to maximise the chances of getting a nice image Incubate the plates for 2-4 days to match colony sizes between plates, then store in fridge until ready to image on the gel doc      FILENAME Yeast spotting assay.doc v1.0 Houseley lab  PAGE 1 AE  / 0 1 2 p y |      #  5 6 N O | n o p r s t u ݻݴᩢjh~9U h)h)h) h%h1 haha haH*h1OJQJ^J h1H*h%OJQJ^Jh1hah%hMh)5hM5CJ(aJ(h%5CJ(aJ(;: ; Z [ # ~  - . | ^gdagd)$a$gd) u v o p q r t v w y z | }  u w x z { } ~ ȷ h)h)h~90JmHnHu h(0Jjh(0JUh%h(mHnHuh%mHnHuh(jh(Ujh~9Uh~9,1h. A!"#$% 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@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w<  EERRRUu    0FMOU!8@0(  B S  ?tvwyz|}tvwyz|}ADEE::py#nttwwADEE::py#ns#"%Mmp.%*Mr2; BcH7TYs,[QycRdgxomu./|fL01(HsFu7bRn()~9datv@tttt@UnknownG.[x Times New Roman5Symbol3. .[x ArialA$BCambria Math"1h0dvgLdvg  !4rr2qHP ?72!xx  Prepare 0jhousele Jon Houseley Oh+'03   @ L Xdlt| Prepare 0 jhouseleNormalJon Houseley4Microsoft Office Word@NZ@H66!@9!G1 RtG   d.@Times New Roman---  2 =xcY  (2 =ceast spotting assay 2 =c.doc  2 =c i 2 =cv1.0   2 =c  2 =PcHouseley   2 =c  2 =clab  2 =c  c''  2 /xc   2 /c1  2 /c  c''@Times New Roman---  2 ycY (2 y-ceast spotting assay         2 yc  @Times New Roman---  2 xc  ---  2 xc   J2 x*cThe day before, leave the plates you will    2 cu 2 cse  2 c  G2 (con the bench overnight (budget 2 plates    (2 xcfor each condition)  2 c    2 xc   2 xVcThis assumes starting with saturated cultures, but it can be done (and probably better     2 c)   +2 xcfrom log phase cells.   2 c    2 xc  @"Arial------------ 2 xcFo  52 cr each strain to be tested,   2 ,cl 2 /cabel   2 Oc4  2 Wcx  2 ^c  .2 cc1.5ml tubes each with 9    2 c0  2 c ---  2 c --- 2 cl water   2 ;c    2 2xc   2 ExcMea "2 Ecsure the OD of   2 E cyour culture  2 E@c    2 Wxc  @Times New Roman--------------- +2 jxcCalculate the volume    2 j crequired   2 j=cf 2 jAcor  2 jNc  2 jTc1x10---  2 esc6---  2 jyc  2 j}ccells  2 jc    2 |xc  ------ 2 x cPut this   2 cv 2 colume   2 c   2 cof culture in  42 3ca 1.5ml tube, dilute to 100  ---  2 c ---  2 cl  2 c  2  cfinal volume    2 9c    2 xc  ------ 2 xcFr  ;2  com this initial dilution, make 4      2 Lc  ,2 Pcserial dilutions of 10    2 c ---  2 c --- 2 cl into  2 c90 ---  2 c --- 2 #cl water    2 Tc   j2 ?cWhen making these dilution series, be sure to vortex each tube      2 = cthoroughly    #2 cbefore pipetting  2  c    2 c    2 xc 0  2 cF   2 co 2 cr  2 ceach  2  cstarting   2 cculture  2 Hcyou s #2 jchould now have 5   2 c  %2 ctubes containing    2 Nc  ---------------  2 xc 0  2 c 0 2 c1x10---  2 c6---  2 c,  2 c  2 c1x10---  2 $c5---  2 *c,  2 .c  2 3c1x10---  2 Rc4---  2 Xc,  2 \c  2 `c1x10---  2 c3---  2 c  2 cand  2 c  2 c1x10---  2 c2---  2 c  2  ccells in 100 ---  2 c ---  2 %cl  2 (c    2 "xc   V2 4x2cPlace your first two plates on a spotting template      2 4c    2 Gxc  ------ 2 YxcSpot 8 ---  2 Yc --- /2 Ycl from each of your 1x10 ---  2 TMc6---  2 YSc  .2 YXccell tubes in the left  2 Yccolumn   2 Yc  2 Y cof each plate  2 Y]c    2 kxc 0 J2 k*cRemember to vortex before taking each spot     2 kc    2 ~xc  --- &2 xcThen spot the 1x10 ---  2 c5---  2 c  G2 (ccell tubes on the next column, and so on    2 c    2 xc   >2 x"cPut the lid on the plate but leave   2 Dc  Y2 K4con the bench for ~1 hour to make sure the liquid is      "2 xcfully absorbed   F2 'cbefore putting upside down in incubator    2 c    2 xc   2 xXcContinue with other plates. It is wise to do 2 replicates of each plate to maximise the        :2 xcchances of getting a nice image    2 =c    2 xc   /2 #xcIncubate the plates for   2 # c2  2 #c- L2 #+c4 days to match colony sizes between plates   #2 #0c, then store in   J2 6x*cfridge until ready to image on the gel doc    2 6c    2 Hxc    2 [xc    2 mxc    2 xc   2 |c  "Systemc--ccbbaa ՜.+,0 hp  University of Edinburgh r  Prepare 0 Title  !"#$%&'()*+,-./012346789:;<?Root Entry Fp9!A1Table WordDocument.SummaryInformation(3DocumentSummaryInformation85CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q