网站地图	本站论坛
高级搜索	收藏本站

热门关键字：　细胞培养　 pcr 　琼脂糖凝胶电泳　质粒DNA的提取　凝胶加样缓冲液　 PCR inventor

	当前位置：试验方案>核酸试验>DNA试验>基因组学> 正文

EMSA using ds Oligonucleotides【Harvard University】

点击： 作者：51protocol收集 来源： 时间： 2007-03-28 　本站论坛

Cepko/Tabin Lab ,Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/E1.html Cepko/Tabin Lab ,Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/E1.html Solutions

10X Annealing Buffer

200 mM Tris 8.0 200 ml 1M Tris pH 8.0ml .5M EDTA pH 8.0ml 5M NaClml Qstore at room temperature

10X Klenow Buffer

500 mM Tris 7.5 500

100 mM MgCl

ml 1M Tris pH 7.52 100 ml 1M MgCl2

10 mM DTT 20

0.5

330

ml 0.5 M DTTmg/ml BSA 50 ml 10 mg/ml BSA (NEB)ml Qstore at -80° C in 50 ml aliq.

2X Binding Buffer (ref: Gutman et al. GENE 110, 1992 pg 197)

20% glycerol 400

20 mM Tris 7.5 20

100 mM KCl 100

1 mM DTT 2

478

ml 50% glycerolml 1M Tris pH 7.5ml 1M KClml 0.5 M DTTml Qmake fresh as needed

Poly dI/dC

Make a 1

Procedure

• Design complementary oligonucleotides with compatible half sites on the ends (I use BamHI and BglII). Dry down 300 ng of the two purified oligos together and resuspend in 9

• Mix the following and incubate at room temperature for 30 min:

dGTP)

Bring up to 200 microliters with TE, phenol/chloroform extract and ethanol precipitate with 0.1 volume of 3 M NaOAc pH 5.2 as well as tRNA. Dry and resuspend in 100

• Generate a table of binding reaction parameters and competitor concentrations. Prepare the gel and running buffer.

EMSA Gel:

8 ml 30% acrylamide (0.8% BIS)

6 ml 50% glycerol

6 ml 10X TBE

40 ml Q

180

Cool to 4° C along with the appropriate amount of 1X TBE.

• Mix the binding reagents in the following order:

i) 10

ii) 3

iii) Q to 20

iv) competitor at 10-20X excess

上一篇：GELSHIFT 下一篇：DNA fingerprinting - agarose gel【California Institute of Technology】