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[DNA Library] Generation of uni-directional nested deletions in plasmid DN 日期：2005-07-18 00:00:00 点击：128 好评：0
Tuesday, November 18, 2003 Description Good Protocol Works well Procedure 1) Digest DNA with two restriction enzymes such that one end is ExoIII susceptible (blunt or 5' overhang) and the other is ExoIII resistant (5' recessed). The ExoIII...
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[DNA Library] Construction of homemade T-vectors 日期：2005-07-18 00:00:00 点击：167 好评：0
Tuesday, November 18, 2003 Description Construction of homemade 'T-vectors' Procedure 1) Digest plasmid to be used with a blunt-end restriction enzyme (I use EcoRV and pBluescript SK-). 2) Clean digestion reaction by phenol/chloroform extr...
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[DNA Library] Bacteriophage Plaque lifts 日期：2005-07-18 00:00:00 点击：215 好评：0
Tuesday, November 18, 2003 Description Plaque transfer is typically performed using Amersham gridded, nylon, Hybond-N membranes. Procedure 1) Place Hybond-N, 82mm membrane circles onto the surface of agar plates carrying bacteriophage infe...
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[DNA Library] Quick Change Mutagenesis 日期：2005-07-18 00:00:00 点击：192 好评：0
Tuesday, November 18, 2003 Description Quick Change Mutagenesis Procedure 1. Set up two separate primer extension reactions (one for each top and bottom primer) containing: 5 microliters 10X Pfu Buffer (supplied with enzyme) 1 microliter 1...
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[DNA Library] S1 nuclease assay using oligonucleotide probe 日期：2005-07-18 00:00:00 点击：187 好评：0
Tuesday, November 18, 2003 Description S1 nuclease assay using oligonucleotide probe Procedure 1. Hybridize RNA and labeled oligonucleotide probe in 50 microliters. Mix the following in an RNAse free 0.2 ml microcentrifuge tube: 15 microgr...
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[DNA Library] EMS Mutagenesis of Yeast 日期：2005-07-18 00:00:00 点击：175 好评：0
Tuesday, November 18, 2003 Description EMS Mutagenesis of Yeast Procedure 1. Grow a 50 ml O/N YPD yeast culture at 30 degrees C. 2. Determine cell density and transfer an amount of culture corresponding to 1 x 108 cells to several 15 ml co...
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[DNA Library] Protocols for shuttle mutagenesis/epitope-tagging of a yeast 日期：2005-07-18 00:00:00 点击：185 好评：0
Tuesday, November 18, 2003 Description Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-lacZ/LEU2 Procedure Shuttle mutagenesis Clone fragment into vector pHSS6 (from strain R1123; map given below). Delete as much...
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[DNA Library] 2 Hybrid system screens 日期：2005-07-18 00:00:00 点击：195 好评：0
Tuesday, November 18, 2003 Description 2 Hybrid system screens Procedure Inoculate the yeast strain containing the first plasmid into the appropriate volume of the appropriate SC-omission medium in a flask and incubate at 30 oC overnight....
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[DNA Library] GST Fusion Protein Purification from Yeast 日期：2005-07-18 00:00:00 点击：165 好评：0
Tuesday, November 18, 2003 Description GST Fusion Protein Purification from Yeast Procedure 5 ml overnight culture of your favorite yeast in your favorite medium. Inoculate 50 ml and grow 30o C shaking O/N until OD600 = 0.8 to 1.2. For SCD...
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[DNA Library] Glass Bead RNA Prep (Yeast) 日期：2005-07-18 00:00:00 点击：165 好评：0
Tuesday, November 18, 2003 Description Glass Bead RNA Prep (Yeast) Procedure 1. Grow cells to no later than late log phase (1E7 cells) in 20 ml of media (protocol can be scaled up or down). 2. Transfer cells to 50 ml Falcon tube. Spin 3000...