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实验技术 > 基础实验技术 > 常用试剂配制

常用试剂配制

最后更新：2007-1-29 阅读次数： 【字体：小 中 大】
TE 1x, 10x
STE 1x, 10x
SSC 20x
Electrophoresis buffers.
TAE 50x
TBE 10x
FGRB 50x
GTGB 20x
TGB 5x
Loading buffer 10x
PEG-NaCl
Bacteriophage lambda buffers
SM
STM
TM
Denaturation buffer
Neutraliszation buffer
Developer Rentgen-2
Fixer BKF-2
Scintillation solution
Plasmid solution I
Plasmid solution II
Oligonucleotide gel staining mix
2M Mg2+ solution
Hybridization buffer HSB (f+)
Restriction buffers
А., B., L., M., H.
Denhardt solution 50х
PBS 10x
SSPE 20x
RNase (DNase free)
Proteinase K solution
EDTA/glycogen solution 20x
DNase RQ1 buffer
Supplement.

TE
1x; pH7.4; 7.6; 8.0; (store at 4oC).
Conc.Stock1ml5ml10ml50ml400ml
Tris Cl10mM1M10µl50µl100µl500µl4ml
EDTA1mM0.5M2µl10µl20µl100µl800µl
H2O mQ988µl4.94ml9.88ml49.4ml395ml
10x:
Conc.Stock1ml5ml
Tris Cl0.1M1M0.1ml0.5ml
EDTA10mM0.5M20µl0.1ml
H2OmQ 880µl4.4ml
sterilize by autoclavingSTE
1x; (store at 4oC).
Conc.Stock1ml5ml10ml50ml
NaCl0.1M5M20µl100µl200µl1.0ml
Tris Cl, pH8.010mM1M10µl50µl100µl0.5ml
EDTA, pH8.01mM0.5M2µl10µl20µl100µl
H2O mQ0.968ml4.84ml9.68ml48.4ml
10x:
Conc.Stock1ml5ml
NaCl1.0M5M0.2ml1.0ml
Tris Cl0.1M1M0.1ml0.5ml
EDTA10mM0.5mM20µl0.1ml
H2OmQ 680µl3.4ml

sterilize by autoclavingSSC
20x; pH 7.0; (store at NT).
Conc.Stock0.5L1.5L2L
NaCl3M58.44g/M87.66g263.0g350.6g
Na3C6H5O7x 5.5H2O0.3M357.1g/M53.57g160.7g214.3g
H2O mQ440.3ml1.32L1.76L
p = 1.103
TAE
1x pH = 7.6:
Tris-acetate 40mM
EDTA 1mM
store the main stock at 4oC, working quantity (~100ml) at NT
Conc.Stock100ml150ml200ml250ml
Tris-base2M121.1g/M24.22g36.33g48.44g60.55g
EDTA0.05M372.3g/M1.862g2.792g3.723g4.654g
Acetic acid~1.56M17.4M~8.96ml
~9.40g		~13.44ml
~14.10g		~17.92ml
~18.80g		~22.40ml
~23.50g		~
~
H2O mQ~73.30g~109.95g~146.60g~183.25g~
p(50x)=1.0878
it is convenient to solubilize Tris and EDTA first then adjust pH by acetic acid. TBE
10x, pH ~8.3;(store at NT in clean bottle)
1x10xStock1L
Tris base89mM0.89M121.14g/M107.8g
Boric acid89mM0.89M61.83g/M55.03g
EDTA, pH 8.02mM20mM500mM40ml
43.84g
H2O  mQ863.3ml

10x stock solution is stable at NT after 0.4µm filtration; for PAAG use as 1x, for agarose gel - as 0.5x; TBE is used in agarose gels for separation of small (< 200bp) fragments. Purification of fragments from TBE-based gels is more complicated than from TAE-based gels. FGRB
50x (formaldehyde gel-running buffer), p=1.104g/ml (store at +4oC):
Conc.Stock0.5L1.0L2.0L
MOPS (pH7.0)1.0M209.27g/M104.64g209.27g418.54g
AcONax3H2O0.5M136.08g/M34.02g68.04g136.08g
EDTAx2H2O50mM372.3g/M9.31g18.62g37.23g
H2O mQ404.0ml808ml1616ml
Conc.Stock0.5L1.0L2.0L
MOPS (pH7.0)1.0M209.27g/M104.64g209.27g418.54g
AcONa anhydrous0.5M82.04g/M20.51g41.02g82.04g
EDTA x 2H2O50mM372.3g/M9.31g18.62g37.23g
H2O mQ417.5ml835ml1670ml

adjust pH7.0 10N NaOH (per 500ml of buffer ~22ml), filter-sterilize through 0.22µm filter; solution becomes yellow during the storage on the light, but it has no effect on the solution quality. GTGB
20x /Glycerol tolerant gel buffer/ (store at +4oC):
Conc.Stock1L1.5L2.0L2.5L
Tris base
1.78M121.14g/M216g324g432g540g
Taurine
0.56M125.15g/M72g108g144g180g
Na2EDTAx2H2O
10.7mM372.3 g/M4g6g8g10g
H2O
 mQ802ml1203ml1604ml2005ml
p=1.094
Taurine: C2H7NO3S; Mw=125.15g/M.TGB
Tris-Glycine buffer 5x, pH8.3, (store the main stock at +4oC, working quantity at NT).
1xConc.Stock1L1.5L
Tris-base25mM125mM121.1g/M15.1g22.71g
Glycine250mM1.25M75.05g/M94g141g
SDS0.1%0.5%10%50ml75ml
H2O  mQ   
Loading buffer
10x, (store at NT).
Conc.Stock1ml5ml10ml25ml
Xilene cyanol0.1%solid1.0mg5.0mg10mg25.0mg
Bromph. blue0.1%solid1.0mg5.0mg10mg25.0mg
SDS0.5%10%50µl250µl500µl1.25ml
EDTA, pH8.00.1M0.5M0.2ml1.0ml2.0ml5.0ml
Glycerol50%100%0.5ml
0.625g		2.5ml
3.125g		5.0ml
6.25g		12.5ml
15.63g
H2O mQ0.25ml1.25ml2.5ml6.25ml
PEG-NaCl
(store at 4oC).
Conc.Stock50ml100ml150ml200ml
NaCl1.6M58.44g/M4.675g9.35g14.03g18.70g
PEG-600013%solid6.50g13.00g19.50g26.00g
or:
Conc.Stock50ml100ml150ml200ml
NaCl1.6M5M16ml
18.97g		32ml
37.95g		48ml
56.92g		64ml
75.90g
PEG-600013%40%16.25ml32.50ml48.75ml65.00ml
SM
(store at 4oC).
Conc.Stock10ml50ml100ml150ml
NaCl0.1M5M200µl1.0ml2.0ml3.0ml
MgSO4*10mM1M100µl0.5ml1.0ml1.5ml
Tris Cl, pH7.550mM1M0.5ml2.5ml5.0ml7.5ml
0.01%2%50µl0.25ml0.5ml0.75ml
H2O mQ9.15ml45.75ml91.5ml137.25ml
* - add after autoclaving;
sterilize by autoclavingSTM
(store at 4oC):
Conc.Stock50ml
NaCl10mM5M100µl
Tris Cl pH8.050mM1M2.5ml
MgCl210mM1M500µl
H2O mQ46.9ml
TM
(store at 4oC):
Conc.Stock50ml
Tris Cl pH8.050mM1M2.5ml
MgCl210mM1M500µl
H2O mQ47.0ml
Denaturation buffer
(store at NT).
Conc.Stock100ml150ml300ml500ml750ml
NaCl1.5M58.44g/M8.766g13.15g26.30g43.83g65.75g
NaOH0.5M10M5ml
6.65g		7.5ml
9.97g		15ml
19.94g		25ml
33.23g		37.5ml
49.84g
  40g/M2g3g6g10g15g
Neutralization buffer
(store at NT).
Conc.Stock150ml300ml500ml750ml
Tris Cl, pH7.41M121.1g/M18.15g36.3g60.5g90.75g
NaCl1.5M58.44g/M13.15g26.30g43.83g65.75g
HCl~0.81M11.6M/L~10.5ml~21ml~35ml~52.5ml
EDTA (optional)1mM0.5M0.3ml0.6ml1ml1.5ml
Developer Rentgen-2
(store at NT in the dark).
500ml800ml1l
1.1g1.76g2.2g
Na2SO3 anhidr.36.0g57.6g72.0g
Hydroquinone4.4g7.04g8.8g
Na2CO324.0g38.4g48.0g
KBr2.0g3.2g4.0g
H2O490ml784ml980ml
solubilize in the presented order , p=1.115g/ml.
Fixer BKF-2
(store at NT).
500ml800ml1l
Na2S2O3x 5H2O130g208g260g
NH4Cl25g40g50g
Sodium pyrosulfite8.5g13.6g17g
H2O398ml636.8ml796ml
solubilize in the presented order, p=1.123g/ml.
Scintillation solution
(store at NT in the dark):
0.5L1L
PPO2.50g5.00g
POPOP0.05g0.10g
Toluene   
Plasmid solution I
(store at +4oC)
Conc.Stock50ml100ml150ml200ml
Glucose50mM2M1.25ml
1.41g		2.5ml
2.825g		3.75ml
4.24g		5.0ml
5.56g
Tris Cl, pH 8.025mM1.0 M1.25ml2.5ml3.75ml5.0ml
EDTA10mM0.5 M1.0ml2.0ml3.0ml4.0ml
H2O mQ46.5ml93ml139.5ml186ml
Plasmid solution II
Conc.Stock0.5ml1ml2.5ml5.0ml6.5ml8.5ml
NaOH0.2N1N0.1ml0.2ml0.5ml1.0ml1.3ml1.7ml
SDS1%10%0.05ml0.1ml0.25ml0.5ml0.65ml0.85ml
H2O mQ0.350ml0.7ml1.75ml3.5ml4.55ml5.95ml
Oligonucleotide gel staining mix
(store at NT).
Conc.Stock100ml200ml300ml500ml
AcONa(5.2)0.5M3M17ml33ml50.0ml83ml
Met. blue0.04%0.2%20ml40ml60ml100ml
H2O mQ73ml127ml190ml317ml
Mg2+2M
(store at NT).
Conc.Stock200ml
MgCl2x6H2O1M203.3g/M40.66g
MgSO4x7H2O1M246.48g/M49.30g
H2O mQ144.6g
p=1.173g/ml.
Hybridization buffer HSB (f+)
high SDS hybridization buffer (store the main stock at 4oC, working - NT)
Conc.Stock500ml
SDS7%solid35g
Formamid50%100%250ml
283.3g
SSC5x20x125ml
145.3g
Bloc. Reagent2%solid10g
NaP (pH 7.0)50mM1M25ml
27.6g
N-Lauroylsarcosine Na0.1%10%5ml
5.1g
Salm.Sperm DNA50µg/ml10µg/µl2.5ml
H2O mQ49ml
p=1.111
Blocking Reagent - is the powder from " http://www.seekbio.com/industry/Special/Roche/Index.shtmlRoche Diagnostics GmBH; former Boehringer Mannheim" #1096176. Instead of 2% Blocking Reagent one may use 10x Denhardt or 2% non-fat dry milk. it is possible to dissolve the Blocking Reagent only in high strength buffer (in this particular case in H2O+SSC+NaP). Salmon sperm DNA should be shredded and denaturated. Restriction buffers 10x
А.
Conc.Stock100µl1ml
Tris Ac (pH7.9 37oC)33mM1M33µl330µl
MgAc10mM1M10µl100µl
KAc66mM5M13.2µl132µl
DTT0.5mM1M0.5µl5µl
H2O mQ43.3µl433µl
B.
Conc.Stock100µl1ml
Tris Cl (pH 8.0 37oC))10mM1M10µl100µl
MgCl25mM1M5µl50µl
NaCl100mM5M20µl200µl
b-Me1mM1M1µl10µl
H2O mQ64µl640µl
L.
Conc.Stock100µl1ml
Tris Cl (pH 7.5 37oC)10mM1M10µl100µl
MgCl210mM1M10µl100µl
DTT1mM1M1µl10µl
H2O mQ79µl790µl
M.
Conc.Stock100µl1ml
Tris Cl (pH7.5 37oC)10mM1M10µl100µl
NaCl50mM5M10µl100µl
MgCl210mM1M10µl100µl
DTT1mM1M1µl10µl
H2O mQ69µl690µl
H.
Conc.Stock100µl1ml
Tris Cl (pH7.5 37oC)50mM1M50µl50µl
MgCl210mM1M10µl100µl
NaCl100mM5M20µl200µl
DTT1mM1M1µl10µl
H20 mQ19µl190µl
Denhardt solution 50x
(store at -20oC)
Conc.Stock50ml300ml1L
Ficoll 4001%(w/v)solid0.5g3.0g10g
Polyvinilpyrolidone1%(w/v)solid0.5g3.0g10g
BSA (fr V)1%(w/v)solid0.5g3.0g10g
H2O mQ46.7ml292.2ml974ml
p=1.004
the solution should be filtered; the solution could be prepared as 100x stock. PBS (recipe 1)
pH(1x)=7.4, (store at NT or at 4oC):
1xConc.Stock100ml500ml
KH2PO41.7mM17mM136.1g/M0.231g1.157g
Na2HPO45.2mM52mM142g/M0.738g3.692g
NaCl150mM1.5M58.44g/M8.77g43.83g
H2O mQ    
1xConc.Stock100ml500ml
KH2PO41.7mM17mM136.1g/M0.231g1.157g
Na2HPO4x7H2O5.2mM52mM268.1g/M1.394g6.971g
NaCl150mM1.5M58.44g/M8.77g43.83g
H2O mQ    
PBS (recipe 2) 10x
pH(1x)=7.3; (store at NT or at 4oC)
1x10xStock100ml500ml
KH2PO41.47mM14.7mM136.1g/M0.2g1.0g
Na2HPO4x7H2O4.29mM42.9mM268.1g/M1.15g5.75g
NaCl137mM1.37M58.44g/M8.0g40.0g
KCl2.68mM26.8mM74.56g/M0.2g1.0g
H2O  mQ   
PBS (recipe 3) 10x
pH(1x)=7.3; (store at NT or at 4oC)
1x10xStock100ml500ml
KH2PO42mM20mM136.1g/M0.272g1.36g
Na2HPO4x7H2O10mM100mM268.1g/M2.68g13.41g
NaCl137mM1.37M58.44g/M8.0g40.0g
KCl2.7mM27mM74.56g/M0.20g1.01g
H2O  mQ   

sterilize by autoclaving; pH is concentration dependent; therefore check pH for diluted 1x buffer only; pH of 10x should be ~6.8 (adjust by NaOH if necessary). SSPE 20x
pH 7.4
Conc.Stock1L
NaCl3M58.44g/M175g
NaH2PO4xH2O200mM137.99g/M27.6g
EDTA20mM372.24g/M7.4g
H2O mQ926ml

adjust pH by 10N NaOH. RNase A (DNase free)
solubilize 10mg/ml in 1-50mM AcONa (pH4.5); incubate 15' in boiling water bath, then cool slowly to NT; store at -20oC, aliquote in use may be stored at 4oC RNAse storage buffer
(store at -20oC).
Conc.Stock1ml5ml10ml
Tris Cl, pH7.610mM1M10µl50µl100µl
Glycerol50%100%0.5ml
0.630g		2.5ml
3.152g		5.0ml
6.305g
H2O mQ0.486g2.43g4.86g
Proteinase K
(store at +4oC, long-term storage at –20oC):
20mg/ml solution in 10mM Tris Cl, 1.0mM CaCl2, 30% glycerol.
EDTA/glycogen solution
20x (store at -20oC):
Conc.Stock1.0ml
EDTA0.2M0.5M400µl
glycogen1mg/ml20mg/ml50µl
H2O mQ550µl
DNase RQ1 buffer
10x (store at -20oC):
Conc.Stock1ml
Tris Cl (pH8.025oC)0.4M1M400µl
NaCl0.1M5M20µl
MgCl260mM1M60µl
CaCl20.1M1M100µl
H20 mQ420µl
DNAse storage buffer
(store at -20oC).
Conc.Stock1ml1.5ml2.5ml5ml10ml
Tris Cl, pH 7.020mM1M20µl30µl50µl100µl200µl
NaCl50mM5M10µl15µl25µl50µl100µl
DTT1mM1M1µl1.5µl2.5µl5.0µl10µl
Glycerol50%100%0.5ml
0.630g		0.75ml
0.9g		1.25ml
1.58g		2.5ml
3.15g		5.0ml
6.31g
H2O mQ0.485ml0.73ml1.21ml2.42ml4.85ml


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