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Thursday, December 04, 2003
Description Digest of YAC-DNA Ligation IPCR with internal primers Procedure Digest of YAC-DNA: YAC-DNA 5 ul 10 X buffer 3 ul restriction enzyme 3 ul H20 21 ul Total 30 ul Incubate at 37 C, 1 hr; heat inactivate at 60 C, 5 min. Restriction enzymes: Alu I Hinc II Rsa I Ligation: * To make sure self ligation occurred try two different DNA concentrations (A & B) for the ligations * If the ligation is inefficient, try cleaning the restriction products by phenol extractions, before starting the ligation- protocol follows For the ligation: A B DNA from digest 5 ul 10 ul 10X digestion buffer 5 ul 5 ul Ligase (5 U) 1 ul 1 ul H20 39 ul 34 ul Total 50 ul 50 ul Incubate at 4 C overnight. IPCR with internal primers PCR-Mix DNA (Ligation mixture) 2 ul dNTP 4 ul 10 X buffer 5 ul Primer -Forward (10uM stock) 1 ul Primer -Reverse (10uM stock) 1 ul Taq 2 units H20 to a total of 50ul IPCR- Program: Step 1 94 C 1.0 min Step 2 94 C 1.0 min Step 3 50 C 1.0 min Step 4 72 C 2.0 min Step 5 30-35 times to Step 2 Step 6 72 C 5.0 min Step 7 4 C forever Separation of IPCR- products in a 1% agarose gel Isolation of amplification fragment (use Gene-Clean or a similar kit) Ligation of the isolated YAC-ends into TA - cloning vector Transformation of ligation mixture into E. coli Checking the transformation for correct YAC-ends insert by PCR PCR-mix: DNA colony 10 X PCR buffer 5.0 ul dNTPs 4.0 ul Primer M13 -for 2.0 ul Primer M13 -rev 2.0 ul Taq 2 units H2O to a total of 50 ul PCR - program as above. Hybridization YAC-ends for checking localization on map Sequence YAC-end plasmid Select PCR primers === ALL OVER AGAIN Phenol Extraction Add equal volume of Phenol Chloroform (25 Phenol: 24 Chloroform: 1 Isoamin alcohol) to the DNA. Spin 10 min at 10,000 rpm. Remove upper phase into clean tubes. Repeat steps 1 and 2. Add equal volume of Chloroform (24:1). Remove upper phase into clean tubes and add 0.5 volume of NH3Act and 3.5 vol. of Ethanol. Incubate at -20 C for 30 min. Spin at 10,000 rpm for 15 min. Discard liquid and dry the pellet. Add 30 ul dd st H2O. Recipes Supplies Tips (责任编辑：泉水) |
YAC library - Generation of YAC-ends by Inverse-PCR (IPCR)
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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