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染色体减数分裂DAPI染色
发表时间:2012-09-14 阅读次数:1660次

 

I. Solutions and Materials(试剂配制)
A. Carnoys Reagent: 60% Ethanol, 30% Chloroform(氯仿), 10% Acetic Acid(醋酸) 
B. 10mM Sodium Citrate(柠檬酸钠) pH 4.5 
C. DAPI Stain: 1 ug/mL DAPI, 50% Glycerol(甘油), 0.1 M Tris pH 9.2, 1 mg/mL Phenylenediamine (二氨次苯基)
E. Cellulase (纤维素酶Sigma C-1184) 
F. Pectolyase (Sigma P-3026) 
G. Cytohelicase (Sigma C-8274)
II. Proceedure (实验步骤)
A. Fix inflorescence in Carnoy's reagent at room temp.: 4 hours with occasional agitation(搅动), 8 hours without agitation(搅动). 
B. Place in -20 for up to a month if you're not going to use immediately 
C. 2x 5 minute Water 
D. 2x 5 minute Sodium Citrate 
E. 2 hours Enzymatic Digestion at 37 degrees in 0.3% Pectolyase, 0.3% cytohelicase, 1% Cellulase. 
F. Wash in ice cold Sodium Citrate 
G. Dissect anthers from buds 
H. Remove excess buffer and add several drops of 60% acetic acid. Macerate(软化) anthers with forcepts. 
I. Stir anthers on 45 degree hotplate for 1 minute. Don't let the slide dry out, Add more acetic acid if necessary. 
J. Flood the slide with ice cold Carnoy's and remove excess liquid. 
K. Squash firmly with coverslip. Dry slide with hair dryer. 
L. Add DAPI stain and visualize under flourescent microscope. 
 
 

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