Solutions for molecular cloning--1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )
Aliquot this into 25 tubes with 500 ul in each tube.
This will keep the nucleotides from being frozed and thawed too much.
To order these nucleotides, call Pharmacia at 1 800-526-3593
and use customer number 6933. Order the dNTP set: 27-2035-01 dNTP set (100mM each dATP, dCTP, dGTP and dTTP- each in 250 ul volume)$174.00
for the set.
20% PEG/2.5 M NaCl:
7.3 g NaCl
10 g PEG (MW=8000) (Fisher P156-3)
Dissolve
in 40 ml double distilled water by stirring, and then adjust the volume to 50 ml.
50% PEG/0.5 M NaCl:
5.85 g NaCl
100 g PEG (MW=8000) (Fisher P156-3)
Dissolve in 100 ml
double distilled water by stirring, and then adjust the volume to 200 ml.
PEG:TE rinse solution: 1:3 solution of 20% PEG containing
2.5M NaCl and 10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA in double distilled water.
250 ul 1 M Tris-HCl, pH 8.0
50 ul 0.5 M EDTA
12.5 ml 20% PEG/2.5 M NaCl.
ddH2O to 37.5 ml
Phenol, TE-saturated: add an equal volume of 10 mM Tris-HCl, pH 7.5-8.0, 1 mM Na2EDTA to ultrapure phenol, mix well,
allow phases to separate, remove and discard upper (aqueous) phase. Repeat until the pH of the aqueous phase is between 7.5-8.0 (store
at 4deg. C).
Phenol/chloroform/isoamyl alcohol (25:25:1):
100 ml TE-saturated phenol
100 ml chloroform
4 ml isoamyl alcohol
204 ml
2M NaOAc (sodium acetate):
27.22 g NaOAc-3H2O
ddH2O to 100 ml
3M NaOAc, pH 4.5:
408.24 g NaOAc-3H2O
Dissolve in approx. 800 ml ddH2O , adjust pH to 4.8 with glacial
acetic acid and bring to a final volume of 1 L with ddH2O.
Restriction enzyme assay buffer, 10X Low Salt: 100 mM Tris-HCl, pH
7.6, 100 mM MgCl2, and 10 mM DTT in sterile double distilled water.
1 ml 1 M Tris-HCl, pH 7.6
1 ml 1 M MgCl2
0.1 ml 1 M DTT
ddH2O to 10 ml
Restriction enzyme assay buffer, 10X Medium Salt: 500 mM NaCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT
in sterile double distilled water.
1.7 ml 3 M NaCl
1 ml 1 M Tris-HCl, pH 7.6
1 ml 1 M MgCl2
0.1 ml 1 M DTT
ddH2O to 10 ml
Restriction enzyme assay buffer, 10X High Salt: 1M NaCl, 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile
double distilled water.
3.3 ml 3 M NaCl
5 ml 1 M Tris-HCl, pH 7.6
1 ml 1 M MgCl2
0.1 ml 1 M DTT
ddH2O to 10 ml
Restriction enzyme assay buffer, 10X SmaI: 200 mM KCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 10 mM DTT in sterile
double distilled water.
2 ml 1 M KCl
1 ml 1 M Tris-HCl, pH 7.6
1 ml 1 M MgCl2
0.1 ml 1 M DTT
ddH2O to 10 ml
RNase T1: 100 U/ul in 50 mM Tris-HCl, pH 7.6
100 ul RNase T1 (Sigma R-8251) (100,000 U/0.2 ml)
25 ul 1 M Tris-HCl, pH 7.6
375 ul ddH2O
500 ul
10% SDS (sodium dodecyl sulfate):
10 g SDS (Fisher S529-3)
ddH2O to 100 ml
1X STB buffer: 25% sucrose and 50 mM Tris-HCl, pH 8.0 in double distilled water.
25 g sucrose
5 ml 1 M Tris-HCl, pH 8.0
ddH2O to 100 ml (filter sterilize and store at 4degC)
Silanizing reagent: 5% solution of dichloro dimethyl silane in 1,1,1-trichloroethane.
Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )
Commonly used solutions (2)--- 1 ) 2 ) 3 )
Solutions for molecular
cloning---1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 7 ) 8 ) 9 )
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