分子遗传学理论与技术基础 Chapter III technology basis(第二部分).ppt

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分子遗传学理论与技术基础 Chapter III technology basis(第二部分).ppt

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分子遗传学理论与技术基础 Chapter III technology basis(第二部分)

④preparation of PAG a: to compound solutions 30%PAG storage solution : Acr 29g, Bis 1g, dH2O add to 100ml ,mixed and lyses keep in brown bottle, at 4℃. TBE buffer(PH8.0)(5x): Tris 54g,Boric acid 27.5g, 0.5M EDTA 20ml, dH2O add to 1000ml , lyses keep at 4℃. 10%AP solution :AP 1g, dH2O add to 10ml , deliquescence(潮解) easily, closed stock, stock at 4℃ only one month. TEMED primary solution: closed stock at 4℃. Gel loading buffer:(鄂P489) formamid 9.5ml, EB 2mg, 0.5M EDTA 520ul,mixed stock at 4℃ . Marker DNA: loading buffer 60ul, TBE(1x) 60ul, phix 174/hacIII,RF DNA 2ul ,mixed in enppondolf tube , stock at 4℃ . b: to wash the appliance of electrophoresis, then drying . (electrophoresis tank gel plate, glass plate etc) C: to do gel casting(凝胶灌制)：鄂P285表14-2 First to compute total volume of PAG solution that is needed in this experiment. preparation of PAG work solution (100ml total volum) PAG work solution(%) 3.5 5.0 8.0 12.0 20.0 30%PAG stock solution 11.6 16.6 26.6 40.0 66.6 ddH2O 67.7 62.7 52.7 39.3 12.7 TBE(5x) 20.0 20.0 20.0 20.0 20.0 10% AP solution 0.7 0.7 0.7 0.7 0.7 To add TEMED 0.005 ml finally ,mixed. Then casting gel solution rapidly, continuously, stay to polymer is formed ready. ⑤electrophoresis process (run gel ) Attention: initiation: to make electrophoresis apparatus, pour electrophoresis buffer ready. first, connect with power, then, turn on electric current . termination: first turn off electric current ,then, break power. ⑥result analysis : ?peel off gel plate . ? stain .