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Wednesday, October 08, 2003
Description Used for cloning of unknown 5-ends (provided internal sequence is available for primer design) Primary Author Larry Simpson ( simpson@hhmi.ucla.edu ) Affiliation University of California Los Angeles , United States Procedure Recipes Purification of extension product cut out the extension product and slice it into small pieces add 100 ul of 0.5 M NH4oAc, 10 mM MgCl; 0.1% SDS incubate with shaking overnight at RT spin down the acrylamide and wash with 100 ul of the same buffer transfer solution in 0.5 ml tube and extract solution with 200 ul of phenol/chl x2 extract with 400 ul of butanol, add 5 mg oh glycogen precipitate with 3V of ethanol at -80 wash the pellet with 70% ethanol dissolve the pellet in 25 ul of water terminal transferase reaction mix 3 ul of 25 mM cobalt solution (supplied with the enzyme, Boehringer) with 2 ul of water; it gives 15 mM 10x stock mix in the following order: 5x buffer 4 ul 10X Co 2 ul 1 mM dGTP 1 ul cDNA in 12 ul TdT, 20 u/ul 1 ul incubate for 30 min at 37 C add 20 ul of water and 1 ul of 10% SDS extract with 50 ul of phenol/chl add 3 ul of 3M NaoAc, pH 7.0 precipitate with 200 ul of ethanol, wash with 100% ethanol, resuspend in 20 ul of water PCR set up PCR in 50 ul using 1 ul of G-tailed cDNA with 0.5 mM of primers (one oligo[C] 15-mer, one- sequence- specific; 0.2 mM of each dNTP; 2.5 mM of MgCl; 94C-30; 65C-30; 72 C-1 min. 35 cycles. Take 5 ul for the gel. If product is not visible or non-specific, take 1 ul for the second PCR. Same conditions. 35 cycles Supplies Tips (责任编辑：泉水) |
5-RACE
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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