常用试剂和培养基文档格式.docx

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常用试剂和培养基文档格式.docx

KH2PO46.0g

NaCl1.0g

NH4Cl2.0g

Add196mlH2O.Autoclave.

Note:

leaveoutNH4Cltoprepare10XM9-N.

10XM9-Nstock(for200ml)

leaveoutNH4Cl!

100XP(for200ml)

KH2PO418.1g

K2HPO45.0g

pHto6.3with10NKOH!

Add192mlH20.Autoclave.

1XSM(for200ml)

NaCl1.16g

MgSO40.20g

Gelatin(2%)1.00mlor20mg

Add190mlH2O,thenadd:

Tris(1M,pH7.5)10.0ml

2%Gelatin(per500ml)

Gelatin10g

Add500mlwater.

20%Succinate(for500ml)

Succinicacid100g

KOH90g

Addto400mlicecoldwater.

MeasurepHto6.4,filtersterilize.

10XTBE(electrophoresisbuffer)

Trisbase108.0g

Na2EDTA9.3g

BoricAcid55.0g

Bringvolumeto1Lwithwater.

Usedoubledistilledwater.

pHshouldbe8.3;

ifnotadjustdropwisewithconc.NaOH.

Wateragarstock(for190ml)

Agar3.0g

Add190mlH2O.

Autoclave,storein60Cwaterbath.

Ragarstock

200ml500ml

Tryptone........2.0g5.0g

NaCl............1.0g2.5g

Agar............3.0g7.5g

AddH2O.........194.0ml485.0ml

LBagar200ml1000ml

Tryptone......2g10g

NaCl..........1g5g

Yeastextract.1g5g

Agar..........3g15g

AddH2O......193ml990ml

Autoclave,storein60Cwaterbath.LBagarcanalsobemadeby

adding10ml10%yeastextractand10ml20XTryptone/NaClto

190mlWA.

NZ200ml500ml

NZamine....2.0g5.0g

NaCl........1.0g2.5g

MgSO4(anhydrous)..0.2g0.5g

Add10MNaOH(1drop/100ml)afteraddingwater.

Foragarmedia:

Agar........2.4g6.0g

Stirbeforeandafterautoclaving.

GYPCtoWAstock:

200ml1000ml

Glucose(40%).....2.0ml10.0ml

Yeastextract(10%)4.0ml20.0ml

100XP............2.0ml10.0ml

CAA(10%).........4.0ml20.0ml

Succinate(20%)....1.0ml5.0ml

Addappropriateantibiotics.

Pourplates.

ORSminimal200ml1000ml

10XM9.........10ml50ml

Succinate(20%).2ml10ml

MSG(10%)......2ml10ml

Glucose(40%)..1ml5ml

MgSO4(0.1M)..1ml5ml

CaCl2(0.5M)..200ul1ml

1000Xvitamins.200ul1ml

Water..........184ml920ml

Omitsuccinateandadd10mlglucoseforstoragemedium.

E.coliminimal200ml1000ml

10XM9..........20ml100ml

MgSO4(0.1M)...2ml10ml

CaCl2(0.5M)...0.2ml1ml

1000Xvitamins..0.2ml1ml

Glucose(40%)...2ml10ml

XCminimal200ml1000ml

10XM9..........5.0ml25ml

MgSO4(0.1M)...1.0ml5ml

200X7aastock..0.2ml1ml

Glucose(40%)...0.5ml2.5ml

Glycerol(50%)..4.0ml20ml

Nifplates

10XNifbuffer100ml

10Xtraceelements100ml

1000Xvitamins1ml

SterileddH2O799ml

Agarose6g

Succinate(20%)20ml

(Succinateisalready0.4%fromthatin10Xnifbuffer)

10XNifbuffer1000ml

K2HPO434.02g

KH2PO457.06g

Succinate(20%)200.00ml

pHshouldbe6.4after10Xdilution.

1000XORSvitamins

Nicotinicacid2.0mg/ml

Pantothenate1.0mg/ml

Biotin0.2mg/ml

Stir,filtersterilize.

Traceelements

MgSO4(0.1M)25ml

CaCl2(0.5M)5ml

Na2MoO4(1mg/ml)5ml

FeCl3(1mg/ml)5ml

Water460ml

YEM(per500ml)

K2HPO40.5g

MgSO47H2O0.2g

NaCl0.1g

Mannitol10.0g

Yeastextract0.4g

Agar15.0g

pHto6.9

YEMsubstitute(per200ml)

To180mlWAstock:

10XM98.0ml

Mannitol(20%)20.0ml

Yeastextract(10%)1.6ml

SM10selectivemedia(forstorage)

10XversatileMg100ml

Thiamine5ml

Threonine5ml

Leucine5ml

Glucose(40%)10ml

Water875ml

Aminoacids

SM10stocks:

Thiamine(0.337%)

Threonine(0.71%)

Leucine(0.79%)

Klebsiella:

Histidine(0.31%)

10XversatileMg(for200ml)

MgSO42.0ml

Thiamine(1mg/ml)2.0ml

CaCl20.2ml

200XPLT(for200ml)

Proline9.20g

Leucine1.60g

Thiamine0.67g

MgPLT

200XPLT5ml

40%glucose10ml

Water885ml

Zbuffer(lacZassays)

200ml1000ml

Na2HPO47H2O....3.22g16.10g

NaH2PO4H2O......1.10g5.50g

KCl..............0.15g0.75g

MgSO4(0.1M)....1.00ml0.20ml

ß

-mercaptoethanol.2.70ml0.54ml

Filtersterilize.

ONPGstock

4mg/mlinphosphatebuffer(pH7)

Phosphatebuffer(pH7.0)per100ml:

Na2HPO47H2O1.64g

NaH2PO4H2O1.38g

0.2MNa2CO3(100ml)

Na2CO3(anhydr.)10.6g

Jensen'

sMedium(Sesbania)

10XMacronutrients(1000ml):

K2HPO43H2O2.6g

NaCl2.0g

MgSO40.97g

CaCl210.0g

FeCl36H2O1.4g

KNO33.0g

1Xsolution(1000ml):

K2HPO43H2O0.20g

NaCl0.20g

MgSO47H2O0.20g

CaHPO41.00g

FeCl36H2O0.10g

orFeEDTA0.26g

KNO3.(onlyfor10X)3.00g

sMedium(1942)

seeVincent1970p.75.

perLiter:

K2HPO43H2O0.2g

NaCl0.2g

MgSO47H2O0.2g

CaHPO41.0g

FeCl36H2O0.1g

orFe-EDTA0.26g

Dilute1:

5whenused.

15mldilutedmedium/pouch.

1000XMicronutrients(200ml)

H3BO3572mg

MnCl2406mg

ZnSO4H2O44mg

CuSO45H2O16mg

NaMO4BH2O18mg

Co2+20mg

Ni2+20mg

Nodulegrindingbuffer(500ml)

50mMKH2PO4(pH7.6),0.15MNaCl

K2HPO4(1M)3.25ml

KH2PO4(1M)21.75ml

NaCl4.38gm

Addto450mldistilledwater,pHto7.6,andadjustvolumeto500ml.

Rapidynegermicide

Forworkingsolution:

Diluteconcentratedstockat0.6ml/L.

1MTris-Cl(500ml)

To400ml,add:

Trisbase60.5g

Addconc.HCltopH

bringtovolume.

TrisbuffermixturesforpH

pHTrisHClTrisbase

7.0.923.077

7.1.905.095

7.2.90.10

7.3.865.135

7.4.84.16

7.5.82.18

7.6.78.22

7.7.74.26

7.8.67.33

7.9.63.37

8.0.50.50

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(~50

ml),adjustto1litervolume.

IV.20XSSC:

175.3gNaCl,88.2gNa3citrate,pHto7.0withconc.HCl(~1

drop),adjustto1litervolume.

Random-primeroligonucleotidelabelling

Mix:

9.00ulDNA(notmorethan200ng)

1.25ulhexamers

Boil,thenplaceonice,thenadd:

5.0ulHEPESbuffer

5.0ulnucleotides[in250mMTris(pH8),25mMMgCl2,50mM

ß

-mercaptoethanol]

1.0ulBSA(40mg/ml)

0.5ulKlenow

5.0ul-32PCTP

Letreactiongoatroomtemperatureforatleast4hours,orovernight.

 

MSMEDIUMFORARABIDOPSIS

To990mlH2Oadd:

Sucrose...........10.0g

MOPS..............0.5g

Agar..............8.0g

AdjustpHto5.7with1MKOH.

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............2.67ml10mM

Nicotinate.........0.4ml10mM

Thiamin............0.02ml10mM

PyridoxineHCl.....0.24ml10mM

Myoinositol........5.5ml100mM

FeSO47H2O........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

Sucrose...........20.0g

KNO3...............26.3ml940mM(95.00g/L)

NaH2PO4H2O........11.0ml100mM

MgSO47H2O.........10.0ml100mM(24.65g/L)

(NH4)2SO3..........10.0ml100mM

CaCl27H2O.........3.3ml300mM(44.00g/L)

Thiamine...........3.0ml10mM

Nicotinate.........0.8ml10mM

PyridoxineHCl.....0.48ml10mM

10XMicronutrients.10.0ml(seebelow)

Sequestrene330Fe.10.0ml(0.28g/100ml)

ForCallusInducingMedium(CIM)add:

2,4-D.....................0.50ml

Kinetin...................0.05ml

ForShootInducingMedium(SIM)add:

IAA.......................0.15ml

6-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)Broth

Bacto-Trypt

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