ࡱ> OQNa (bjbj .,hGbhGb>O44444HHH8,H(!######$dGE4G44QQQ44!Q!QQQg~YvQ 0QQ4QQGGQB : Synchronised Meiosis in Budding Yeast These experiments are usually performed in the SK1 strain. This has a few unusual features compared to other common budding yeast lab strains: These cells clump together (flocculate) very readily this means that on plates they have a strange crusty consistency, while in liquid they form large particles. To take an OD measurement, vortex culture well and pipette into cuvette, take reading immediately. Do not set up a number of cuvettes and then take readings as cells will start to settle distorting the results. They sporulate as soon as they stop growing. Having selected diploids, freeze them and then follow the protocols below to avoid haploid contamination. Transformations must be performed at OD<1 for good efficiency They become petite (loose mitochondrial DNA) very easily. Before using in an experiment, grow on YPglycerol at least once. Deletion of markers was done using hisG recombination, leaving a hisG mark. Using different auxotrophic markers can alter sporulation rate, so always compare cells with the same set of auxotrophies. To purify SK1 diploids: Put a small amount of the ( strain on a YPD plate. Mix with an excess of a cells, and also put an equivalent amount of a cells in a separate patch on the plate. Grow cells for a few hours at 30( Make 2x 1(l spots of 5mg/ml (-factor on a YPD plate and allow to dry. Mark the sites precisely as you cannot see them once dried. Pick a small amount cells from the centre of the patch of a cells, put into 100l water and vortex well. Spot 1l of this on one of the patches of -factor. Repeat for the patch of mated cells. Incubate the plate O/N at 25( Next day, there should be clear growth on the patch of -factor with the mated cells, but much less on the patch of only a. If the difference is not clear, make two more spots of -factor and spot on cells from the first round of selection as above. Inoculate cells into 4ml YPD, grow O/N at 30( First thing next morning, mix 500(l overnight culture with 500(l sterile 30% glycerol and freeze. Store this glycerol stock at -80(. It is wise to make multiple glycerol stocks of each strain, as you will need to use it for every experiment. Synchronised meiosis: Monday morning: Patch diploid cells from glycerol stocks on a YP glycerol plate, O/N at 30( Tuesday morning: Patch these cells onto 4% YPD plate, leave at 30( (can leave these growing overnight and inoculate the next evening if desired). Tuesday evening: Inoculate cells into 5ml YPD Wednesday evening: Inoculate 20ml YPA with cells from the YPD overnight to OD 0.2 (for growth at 30() or 0.5 (for growth at 25(). Leave shaking overnight. Thursday morning: Cells should be ~OD 1.8-1.9 (4-4.5x107 cells/ml). If slightly overgrown, dilute cultures to this OD. Spin down cells, re-suspend in the same volume of SPO media, spin, pour off and re-suspend again in the same volume of SPO media. Shake cultures at desired sporulation temperature FAST (250rpm) DNA replication should take place over 5-6 hours depending on temperature, rapidly followed by recombination. Monitor replication by FACS and recombination by PFGE. By the next morning most cells should form tetrads. Notes: Always start from the glycerol stock, and do not stop the protocol over the weekend at any point. SK1 diploids left on plates in the fridge will sporulate, which will screw up the meiosis course. This protocol is from Adele Marston If meiosis goes too fast, slow down the shaking speed slightly (230rpm) Media 4% YPD plates: normal YPD with double the glucose YP glycerol plates: YP plates with 2% v/v glycerol Autoclave the plates minimally; they can be destroyed by over-cooking YPA: 20g peptone 10g yeast extract 20g KOAc Dissolve in 1000ml total volume Filter sterilise into 2 bottles SPO: 3g KOAc 5mg uracil 5mg histidine 25mg leucine 12.5 tryptophan Dissolve in 1L total volume Add 1ml filter sterilised 20% raffinose Test pH using paper, add a few l of acetic acid to pH ~7 if needed Filter sterilise into 2 bottles %&Z [ \  0    : @ I N O P h i + , - 7 8 : C J K R S b r ۻǰǫǫǤǖےۋǒے jah]XhhK jmh]X h!h! jh! h!5 jah}h!5h]Xh]X5hG)h!hHh}hSh{p"h]XhLh5h5h{5CJ(aJ(h5h55CJ(aJ(6&'\ ] 0 1   N O P h i - . gd]X^gdL^gd]Xgd}$a$gd5. "$vx"#4^gd{p"gd{p"^gd]Xgd]X  $Frt >? !"de JKef .EseŽ干ʹh!xh!x5h!xhEih}hct hW<H*hW< jh{p"h{p"h}h]X5 hF25hF2 jmh]XhQ h!h! h!5h]X jh]Xh!h!5h!hhK9hiEFgd!x^gdW<gd{p"^gd{p"Qao(=(>(?(A(B(D(E(G(H(J(K(U(V(j(k(p(y(}((((((((((𿹿h]0JmHnHu hi 0Jjhi 0JUhl|hi mHnHujhi Uh]jh]Uh{p"Uhi h-zhOh!x+ 0?Qo(<(=(>(@(A(C(D(F(G(I(J(~((^gdOgdOgd{p"gd!xgd-z This is 0.3% KOAc, 0.02% raffinose, amino acids at 0.25x      FILENAME Synchronised meiosis v1.8 Houseley lab  PAGE 1 (((((gd{p",1h. A!"#$% 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 wHeader  9r 4 @4 wFooter  9r .)@. w Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< %, AANNNQ ( . (( ,BIKQ!8@0(  B S  ?6?is?K5 > >@ACDFGIJVW  ';>33330<=>>AAJ}ih-EiwG)N 50S/i {p"?k#b%^&4I*F+y,40F2Mc2%4A5wu56xx>hKZMf;NnPwPS]XY:\e. iiisctUvOvTxy[y-zY=z{B]` LFL\w! |OQ(K~eMrHW<!xY c <;O>Lr]QYYVRGL^}^#W[|9`rI!QS+3`_S-)Ukutl|P>@@====@(@((($@(P@UnknownG.[x Times New Roman5Symbol3. .[x ArialA$BCambria Math"1h,FJzg#gl l !4662Q HP?52!xxU,F Jonathan Houseley Jon Houseley Oh+'0<  4 @ L Xdlt|Jonathan HouseleyNormalJon Houseley17Microsoft Office Word@ ~@#@ @p2~l G ; Rt+ u  d.@Times New Roman--- )2 =xcSynchronised meiosis     2 =c { 2 =cv1.8   2 =c  2 =PcHouseley   2 =c  2 =clab  2 =c  c''  2 /xc   2 /c1  2 /c  c''@Times New Roman--- C2 y%cSynchronised Meiosis in Budding Yeast            2 yoc  ---  2 xc   2 xRcThese experiments are usually performed in the SK1 strain. This has a few unusual           +2 xcfeatures compared to   2  cother common   2 [c  22 ccbudding yeast lab strains:   2 c    2 xc   [2 x5cThese cells clump together (flocculate) very readily      2 c  2 c  &2 cthis means that on   2 Fc  2 K cplates they   2 xMchave a strange crusty consistency, while in liquid they form large particles.      2 Sc   2 \c    2 c   2 xVcTo take an OD measurement, vortex culture well and pipette into cuvette, take reading        2 xMcimmediately. Do not set up a number of cuvettes and then take readings as cel     2 jcls will     "2 2xcstart to settle  2 2c  .2 2cdistorting the results.   2 2Qc    2 Exc   2 WxScThey sporulate as soon as they stop growing. Having selected diploids, freeze them       82 ixcand then follow the protocols   2 i8cbelow   2 iccto av 22 icoid haploid contamination.    2 i)c    2 |c   ^2 x7cTransformations must be performed at OD<1 for good effi       2 cciency  2 c    2 xc   2 xcThey  2 cbecome   2 c  D2 &cpetite (loose mitochondrial DNA) very     2 ceasily 2  c. Before   2 - cusing in an   P2 x.cexperiment, grow on YPglycerol at least once.         2 c    2 xc   |2 xKcDeletion of markers was done using hisG recombination, leaving a hisG mark.              2 mc    2 xc   P2 x.cUsing different auxotrophic markers can alter    A2 $csporulation rate, so always compare       I2 !x)ccells with the same set of auxotrophies.      2 !xc    2 4xc    2 Fc  @Times New Roman--- .2 YxcTo purify SK1 diploids:    2 Yc  ---  2 kxc  @Symbol--------------- 22 xcPut a small amount of the    ---  2  ca ---  2 ,c  O2 /-cstrain on a YPD plate. Mix with an excess of   ---  2 Qca---  2 Yc  2 ] ccells, and  --- =2 x!calso put an equivalent amount of   ---  2 Mca---  2 Uc  82 Yccells in a separate patch on t 2   che plate.  2 Bc    2 xc  @Symbol------------ ;2 x cGrow cells for a few hours at 30   ---  2 Gc---  2 Nc    2 xc  --------------- 2 x cMake 2x 1---  2 cm --- 2 cl  2  cspots of  2 c5mg/ml    ---  2 :ca ---  2 Ec- 2 Kcfactor  2 nc  "2 scon a YPD plate   #2 cand allow to dry  2 @ c. Mark the   V2 x2csites precisely as you cannot see them once dried.    2 c    2 xc  --- b2 x:cPick a small amount cells from the centre of the patch of    ---  2 ca---  2 c  (2 ccells, put into 100 2 e cl water    'x@Times New Roman--.e2 'x<cand vortex well. Spot 1l of this on one of the patches of   .-  2 'c- .2 'cfactor. Repeat for the   ------ V2 ;x2cpatch of mated cells. Incubate the plate O/N at 25   ---  2 ;c---  2 ;c    2 Mxc   _x@Times New Roman--._2 _x8cNext day, there should be clear growth on the patch of  .-  2 _c- 72 _cfactor with the mated cells,    --- /2 qxcbut much less on the pat  2 q cch of only ---  2 qXca--- S2 q`0c. If the difference is not clear, make two more       x@Times New Roman--.2 x cspots of .-  2 c- q2 Dcfactor and spot on cells from the first round of selection as above.   2 _c    2 xc  ------ ,2 xcInoculate cells into 4  ,2 cml YPD, grow O/N at 30     ---  2 c---  2 c    2 xc  ------ =2 x!cFirst thing next morning, mix 500    ---  2 Qcm --- 52 Zcl overnight culture with 500  ---  2  cm --- 2 cl ste 2 0 crile 30% g  2 rclycerol    --- 2 x cand freeze :2 c. Store this glycerol stock at      2 qc- 2 wc80---  2 c--- F2 'c. It is wise to make multiple glycerol         v2 xGcstocks of each strain, as you will need to use it for every experiment.     2 +c   @Times New Roman--@Times New Roman--- "Systemd4- B#--ccbbaaTV ՜.+,0 hp  Edinburgh University6  Title  !"#$%&()*+,-./0123456789:;<=>?@ABCDEGHIJKLMPRoot Entry F7j~R1TableWordDocument.,SummaryInformation('<DocumentSummaryInformation8FCompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q