DNA操作试剂配方
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1.5×CTAB
CTAB 15 g
1 M Tris·Cl (pH 8.0) 75 ml
0.5 M EDTA 30 ml
NaCl 61.4 g
add ddH2O to 1000 ml
0.5 M EDTA (pH 8.0)
EDTA-Na·2H2O 186.1 g
NaOH ~20 g
Adjust to pH 8.0
dH2O to 1000 ml
sterilize by autoclaving
1 M Tris·HCl
pH 7.4 pH 7.6 pH 8.0
Tris base 121.1 g 121.1 g 121.1 g
Concentracted HCl ~70 ml ~64 ml ~42 ml
dH2O to 1000 ml 1000 ml 1000 ml
Sterilize by autoclaving
TE (pH 8.0)
Stock vol.
10 mM Tris·HCl (pH 8.0) 1 M 10 ml
1 mM EDTA (pH 8.0) 0.5 M 2 ml
dH2O to 1000 ml
sterilize by autoclaving
10 M NH4Ac
NH4Ac 385 g 770 g
H2O to 500 ml 1000 ml
10×PCR buffer
stock vol.
500 mM KCl 2.5 M(sterilized) 200 ml
100 mM Tris-HCl 1 M pH 9.0(sterilized) 100 ml
1% Triton X-100 100% 10 ml
ddH2O 690 ml
sterilize by autoclaving
5×TBE
Tris 54 g
Boric acid 27.5 g
0.5 M EDTA (pH 7.9) 20 ml
dH2O to 1000 ml
10×TAE
Tris 121.1 g 484.4 g
EDTA(0.5 M) 20 ml 80 ml
NaAc·3H2O 17 g 68 g
glacial acetic acid ~30 ml ~200 ml
adjust to pH 8.1
dH2O to 1000ml 4000ml
NaOH
10 N 4 N
NaOH 400 g 160 g
dH2O to 1000 ml 1000 ml
2 N HCl
concentrated HCl 365 ml 182.5 ml
dH2O to 2000 ml 1000 ml
5 mg/ml ssDNA
Salmon sperm DNA 1 g
ddH2O to 200 ml
0.5 M P.B (phosphate Buffer) pH 6.8
Na2HPO4 16.44 g 131.52 g
NaH2PO4 16.11 g 128.88 g
dH2O to 500 ml 4000 ml
20×SSC
NaCl 175.3 g 701.2 g
Na3Citrate 88.2 g 352.8 g
dH2O to 1000 ml 4000 ml
Sterilize by autoclaving
10% SDS
SDS 100 g
dH2O to 1000 ml
Heat to 68 ℃ to assist dissolution
50×Denhart’s Solution
Ficoll 400 10 g
PVP-360 10 g
BSA (Fraction V) 10 g
ddH2O to 1000 ml
Southern Blot Hybridization Buffer (Saghai,s Lab)
Final conc. Stock Vol.
5×SSC 20× 250 ml
50 mM PB (pH 6.8) 0.5 M 100 ml
5×Denhardt’s 50× 100 ml
2.5 mM EDTA (pH 8.0) 0.5 M 5 ml
100 μg/ml ssDNA 5 mg/ml 20 ml
0.4%SDS 20% 20 ml
Dextran sulfate 50 g
ddH2O to 1000 ml
(Place a beaker on a stirrer, add these solution in the order of appearance one by one. SDS should be the very last item.)
Washing off Probe for Re-hybridization of Blots (I)
Washing time: 10 min
Final conc. Stock Vol.
0.1×SSC 20×SSC 20 ml
0.1% SDS 10% SDS 40 ml
dH2O to 4000 ml
Washing off Probe for Re-hybridization of Blots (II)
Washing time: 3 min
Final conc. Stock Vol.
0.1 N NaOH 10 N NaOH 40 ml
0.2% SDS 10% SDS 80 ml
dH2O to 4000 ml
Washing off Probe for Re-hybridization of Blots(Ⅲ)
Washing time: 20 min
Final conc. Stock Vol.
0.2 M Tris. (pH 7.5) 1 M Tris. (pH 7.5) 800 ml
0.1×SSC 20×SSC 20 ml
0.2% SDS 10% SDS 80ml
dH2O to 4000ml
Blue Juice
Final conc. Stock Vol. Vol.
70% Glycerol 100% 35 ml 70 ml
0.5×TBE 5× 5 ml 10 ml
0.2% SDS 10% 1 ml 2 ml
20 mM EDTA 0.5 M 2 ml 4 ml
5 mg/ml Bromphenol Blue 0.25 g 0.5 g
5 mg/ml Xylene cyanol 0.25 g 0.5 g
dH2O to 50 ml 100 ml
EB (10 mg/ml)
ehidium bromide 1 g
dH2O to 100 ml .Stir on a magnetic stirrer for several hours. Transfer the solution to a dark bottle and store at 4 ℃. The concentration of work solution: 0.5 μg/μl (50 μl stock solution In 1000 ml dH2O).
Decontamination of EB
Reduce the concentration of EB <0.5 mg/ml, add 1 volume of 0.5 M KMnO4,mix carefully then add 1 volume of 2.5 N HCl,mix carefully and allow the solution to stand at room temperature for several hours. Add 1 volume of 2.5 N NaOH, mix and discard.
