士锋DNA操作试剂配方

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 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 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.