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常用试剂和培养基

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Sterilewaterbottles

200ml,500ml,or950mlwater.Autoclave.

0.05MCaCl₂(per200ml)

CaCl₂·2H₂O1.5gAdd198mlwater.Autoclave.

0.5MCaCl₂(per200ml)

CaCl₂·2H₂O15.0gAdd188mlwater.Autoclave.

0.1MMgSO₄(per200ml)

MgSO₄(anhydrous)2.4gAdd200mlwater.Autoclave.

10%MSG(for200ml)

Glutamicacid(Na)20.0gAdd188mlwater.Autoclave.

40%Glucose(per200ml)

Glucose(dextrose)80.0gAddwaterto200ml(~145-150ml).Autoclave.

10%CAA(per200ml)

CaseinAcidHydrolysate20.0gAdd186mlwater.Autoclave.

10%Yeast(per200ml)

Yeastextract20.0gAdd150-160mlwater.Autoclave.Add150ulß-mercaptoethanolafterautoclaving.

20XTryptone/NaCl(per200ml)

Tryptone40.0gNaCl20.0gAdd190mlwater.Autoclave.Add150ulß-mercaptoethanolafterautoclaving.

10XM9stock(for200ml)

Na₂HPO₄(anhydr.)12.0gKH₂PO₄6.0gNaCl1.0gNH₄Cl2.0gAdd196mlH₂O.Autoclave.Note:leaveoutNH₄Cltoprepare10XM9-N.

10XM9-Nstock(for200ml)

Na₂HPO₄(anhydr.)12.0gKH₂PO₄6.0gNaCl1.0gAdd196mlH₂O.Autoclave.Note:leaveoutNH₄Cl!

100XP(for200ml)

KH₂PO₄18.1gK₂HPO₄5.0gpHto6.3with10NKOH!Add192mlH₂0.Autoclave.

1XSM(for200ml)

NaCl1.16gMgSO₄0.20gGelatin(2%)1.00mlor20mgAdd190mlH₂O,thenadd:Tris(1M,pH7.5)10.0mlAutoclave.

2%Gelatin(per500ml)

Gelatin10gAdd500mlwater.Autoclave.

20%Succinate(for500ml)

Succinicacid100gKOH90gAddto400mlicecoldwater.MeasurepHto6.4,filtersterilize.

10XTBE(electrophoresisbuffer)

Trisbase108.0gNa₂EDTA9.3gBoricAcid55.0gBringvolumeto1Lwithwater.Usedoubledistilledwater.pHshouldbe8.3;ifnotadjustdropwisewithconc.NaOH.

Wateragarstock(for190ml)

Agar3.0gAdd190mlH₂O.Autoclave,storein60Cwaterbath.

Ragarstock

200ml500mlTryptone........2.0g5.0gNaCl............1.0g2.5gAgar............3.0g7.5gAddH₂O.........194.0ml485.0mlAutoclave,storein60Cwaterbath.

LBagar200ml1000ml

Tryptone......2g10gNaCl..........1g5gYeastextract.1g5gAgar..........3g15gAddH₂O......193ml990mlAutoclave,storein60Cwaterbath.LBagarcanalsobemadebyadding10ml10%yeastextractand10ml20XTryptone/NaClto190mlWA.

NZ200ml500ml

NZamine....2.0g5.0gNaCl........1.0g2.5gMgSO₄(anhydrous)..0.2g0.5gAdd10MNaOH(1drop/100ml)afteraddingwater.Autoclave.Foragarmedia:Agar........2.4g6.0gStirbeforeandafterautoclaving.

GYPCtoWAstock:200ml1000ml

Glucose(40%).....2.0ml10.0mlYeastextract(10%)4.0ml20.0ml100XP............2.0ml10.0mlCAA(10%).........4.0ml20.0mlSuccinate(20%)....1.0ml5.0mlAddappropriateantibiotics.Pourplates.

ORSminimal200ml1000ml

10XM9.........10ml50mlSuccinate(20%).2ml10mlMSG(10%)......2ml10mlGlucose(40%)..1ml5mlMgSO₄(0.1M)..1ml5mlCaCl₂(0.5M)..200ul1ml1000Xvitamins.200ul1mlWater..........184ml920mlOmitsuccinateandadd10mlglucoseforstoragemedium.

E.coliminimal200ml1000ml

10XM9..........20ml100mlMgSO₄(0.1M)...2ml10mlCaCl₂(0.5M)...0.2ml1ml1000Xvitamins..0.2ml1mlGlucose(40%)...2ml10ml

XCminimal200ml1000ml

10XM9..........5.0ml25mlMgSO₄(0.1M)...1.0ml5mlCaCl₂(0.5M)...0.2ml1ml200X7aastock..0.2ml1mlGlucose(40%)...0.5ml2.5mlGlycerol(50%)..4.0ml20ml

Nifplates

10XNifbuffer100ml10Xtraceelements100ml1000Xvitamins1mlSterileddH₂O799mlAgarose6gSuccinate(20%)20ml(Succinateisalready0.4%fromthatin10Xnifbuffer)

10XNifbuffer1000ml

K₂HPO₄34.02gKH₂PO₄57.06gSuccinate(20%)200.00mlpHshouldbe6.4after10Xdilution.

1000XORSvitamins

Nicotinicacid2.0mg/mlPantothenate1.0mg/mlBiotin0.2mg/mlStir,filtersterilize.

Traceelements

MgSO₄(0.1M)25mlCaCl₂(0.5M)5mlNa₂MoO₄(1mg/ml)5mlFeCl₃(1mg/ml)5mlWater460ml

YEM(per500ml)

K₂HPO₄0.5gMgSO₄7H₂O0.2gNaCl0.1gMannitol10.0gYeastextract0.4gAgar15.0gpHto6.9

YEMsubstitute(per200ml)

To180mlWAstock:10XM98.0mlMannitol(20%)20.0mlYeastextract(10%)1.6ml

SM10selectivemedia(forstorage)

10XversatileMg100mlThiamine5mlThreonine5mlLeucine5mlGlucose(40%)10mlWater875ml

Aminoacids

SM10stocks:Thiamine(0.337%)Threonine(0.71%)Leucine(0.79%)Klebsiella:Histidine(0.31%)

10XversatileMg(for200ml)

MgSO₄2.0mlThiamine(1mg/ml)2.0mlCaCl₂0.2ml

200XPLT(for200ml)

Proline9.20gLeucine1.60gThiamine0.67gAutoclave.

MgPLT

10XversatileMg100ml200XPLT5ml40%glucose10mlWater885ml

Zbuffer(lacZassays)

200ml1000mlNa₂HPO₄7H₂O....3.22g16.10gNaH₂PO₄H₂O......1.10g5.50gKCl..............0.15g0.75gMgSO₄(0.1M)....1.00ml0.20mlß-mercaptoethanol.2.70ml0.54mlFiltersterilize.

ONPGstock

4mg/mlinphosphatebuffer(pH7)

Phosphatebuffer(pH7.0)per100ml:

Na₂HPO₄7H₂O1.64gNaH₂PO₄H₂O1.38g

0.2MNa₂CO₃(100ml)

Na₂CO₃(anhydr.)10.6g

Jensen"sMedium(Sesbania)

10XMacronutrients(1000ml):K₂HPO₄3H₂O2.6gNaCl2.0gMgSO₄0.97gCaCl₂10.0gFeCl₃6H₂O1.4gKNO₃3.0g

Jensen"sMedium(Sesbania)

1Xsolution(1000ml):K₂HPO₄3H₂O0.20gNaCl0.20gMgSO₄7H₂O0.20gCaHPO₄1.00gFeCl₃6H₂O0.10gorFeEDTA0.26gKNO₃.(onlyfor10X)3.00g

Jensen"sMedium(1942)

seeVincent1970p.75.perLiter:K₂HPO₄3H₂O0.2gNaCl0.2gMgSO₄7H₂O0.2gCaHPO₄1.0gFeCl₃6H₂O0.1gorFe-EDTA0.26gDilute1:5whenused.15mldilutedmedium/pouch.

1000XMicronutrients(200ml)

H₃BO₃572mgMnCl₂406mgZnSO₄H₂O44mgCuSO₄5H₂O16mgNaMO₄BH₂O18mgCo2+20mgNi2+20mg

Nodulegrindingbuffer(500ml)50mMKH₂PO₄(pH7.6),0.15MNaCl

K₂HPO₄(1M)3.25mlKH₂PO₄(1M)21.75mlNaCl4.38gmAddto450mldistilledwater,pHto7.6,andadjustvolumeto500ml.

Rapidynegermicide

Forworkingsolution:Diluteconcentratedstockat0.6ml/L.

1MTris-Cl(500ml)

To400ml,add:Trisbase60.5gAddconc.HCltopHbringtovolume.

TrisbuffermixturesforpH

pHTrisHClTrisbase7.0.923.0777.1.905.0957.2.90.107.3.865.1357.4.84.167.5.82.187.6.78.227.7.74.267.8.67.337.9.63.378.0.50.50

SouthernTransfersolutions

I.0.25NHCl:21.5mlconc.HCl(11.6N)to1litervolume.II.0.5MNaOH,1.5MNaCl:87.7gNaCl,20.0gNaOHto1litervolume.III.1.0MTris,1.5MNaCl:121.1gTrisbase,87.7gNaCl,pHto8.0withconc.HCl(~50ml),adjustto1litervolume.IV.20XSSC:175.3gNaCl,88.2gNa3citrate,pHto7.0withconc.HCl(~1drop),adjustto1litervolume.

Random-primeroligonucleotidelabelling

Mix:9.00ulDNA(notmorethan200ng)1.25ulhexamersBoil,thenplaceonice,thenadd:5.0ulHEPESbuffer5.0ulnucleotides[in250mMTris(pH8),25mMMgCl₂,50mMß-mercaptoethanol]1.0ulBSA(40mg/ml)0.5ulKlenow5.0ul-32PCTPLetreactiongoatroomtemperatureforatleast4hours,orovernight.
MSMEDIUMFORARABIDOPSIS
To990mlH₂Oadd:Sucrose...........10.0gMOPS..............0.5gAgar..............8.0gAdjustpHto5.7with1MKOH.Autoclave.Whencooledaddsterilestocksolutionsof:KNO₃...............20.0ml940mM(95.00g/L)NH₄NO₃.............20.0ml1000mM(80.04g/L)KH₂PO₄.............12.5ml100mM(13.60g/L)MgSO₄7H₂O.........15.0ml100mM(24.65g/L)CaCl₂7H₂O.........10.0ml300mM(44.00g/L)Glycine............2.67ml10mMNicotinate.........0.4ml10mMThiamin............0.02ml10mMPyridoxineHCl.....0.24ml10mMMyoinositol........5.5ml100mMFeSO₄7H₂O........1.0ml(2.780g/100ml)Na₂EDTA...........1.0ml100mM(3.720g/100ml)MnSO₄4H₂O.........1.0ml100mM(1.690g/100ml)ZnSO₄4H₂O.........3.0ml10mM(0.288g/100ml)H₃BO₃.............1.0ml100mM(0.618g/100ml)KI.................0.5ml10mM(0.166g/100ml)CuSO₄5H₂O........0.01ml10mM(0.170g/100ml)Na₂MoO₄2H₂O......0.1ml10mM(0.242g/100ml)CoCl₂6H₂O........0.01ml10mM(0.238g/100ml)
B5MEDIUMFORARABIDOPSIS
To990mlH₂Oadd:Sucrose...........20.0gMOPS..............0.5gAgar..............8.0gAdjustpHto5.7with1MKOH.Autoclave.Whencooledaddsterilestocksolutionsof:KNO₃...............26.3ml940mM(95.00g/L)NaH₂PO₄H₂O........11.0ml100mMMgSO₄7H₂O.........10.0ml100mM(24.65g/L)(NH₄)2SO3..........10.0ml100mMCaCl₂7H₂O.........3.3ml300mM(44.00g/L)Myoinositol........5.5ml100mMThiamine...........3.0ml10mMNicotinate.........0.8ml10mMPyridoxineHCl.....0.48ml10mM10XMicronutrients.10.0ml(seebelow)Sequestrene330Fe.10.0ml(0.28g/100ml)ForCallusInducingMedium(CIM)add:2,4-D.....................0.50mlKinetin...................0.05mlForShootInducingMedium(SIM)add:IAA.......................0.15ml6-dimethylaminopurine.....5.00ml
10XMicronutrients(per100ml)MnSO₄4H₂O.........0.6ml100mM(1.690g/100ml)ZnSO₄4H₂O.........10.07ml10mM(0.288g/100ml)H₃BO₃.............4.9ml100mM(0.618g/100ml)KI.................4.5ml10mM(0.166g/100ml)CuSO₄5H₂O........2.3ml10mM(0.170g/100ml)Na₂MoO₄2H₂O......1.0ml10mM(0.242g/100ml)CoCl₂6H₂O........1.0ml10mM(0.238g/100ml)
LB(Luria-Bertani)BrothBacto-Tryptone10gBacto-YeastExtract5gNaCl5gH₂Oupto1LIfdesired,adjustthepH7.5withNaOH.
CA(CompleteAgar)CaseinHydrolysate10gYeastExtract5gK2HPO₄4gAgar15gH₂Oupto1L
StabAgarNutrientBroth10gNaCl5gAgar6gH₂Oupto1LMixwellandautoclavefor15minat121C.
S.O.C.(fromBRL)1X100ml2%Bacto-Tryptone2.0g0.5%YeastExtract0.5g10mMNaCl2.92g2.5mMKCl3.73g20mMMgCl₂20.2g20mMMgSO₄24.6g20mMGlucose9.0gMixabove,bringto100ml,andfilterthroughasterile0.45µmfilter.Storethestocksolutionat-20Cor-70C.
BacillusThuringiensisMediumComponentG/LGlucose3.0(NH4)₂SO₄2.0YeastExtract2.0K2HPO₄3H₂O0.5MgSO₄*7H₂O0.2CaCl₂*2H₂O0.08MnSO₄*4H₂O0.05Addcomponenetstodistilledwater.MixthroughlyandbringpHto7.3.Autoclavefor15minat15psi--121°C