-
[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...
-
[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...
-
[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...
-
[DNA Library] Protocols for shuttle mutagenesis/epitope-tagging of a yeast 日期：2005-07-18 00:00:00 点击：184 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...
-
[DNA Library] 2 Hybrid system screens 日期：2005-07-18 00:00:00 点击：194 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....
-
[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...
-
[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...
-
[DNA Library] Phenol/Freeze RNA Prep (Yeast) 日期：2005-07-18 00:00:00 点击：159 好评：0
Tuesday, November 18, 2003 Description Phenol/Freeze RNA Prep (Yeast) Procedure 1. Grow 10 ml of yeast to 5E6 cells/ml. 2. Harvest cells by centrifugation and resuspend cells in 400 l of AE buffer. 3. Transfer cells to a 1.5 ml microcentri...
-
[DNA Library] Gene Disruption via PCR (Yeast) 日期：2005-07-18 00:00:00 点击：191 好评：0
Tuesday, November 18, 2003 Description Gene Disruption via PCR (Yeast) Procedure Reaction mix: 5 l 10X Taq Buffer 5 l 25 mM MgCl2 2 l 10 mM dNTP's 10-100 ng template DNA 25 pmols of each primer 0.5 l Taq polymerase (2 Units) ______________...
-
[DNA Library] Electroporation (Yeast) 日期：2005-07-18 00:00:00 点击：167 好评：0
Tuesday, November 18, 2003 Description Electroporation (Yeast) Procedure 1. Inoculate 50ml YEPD with a colony and grow with shaking at 30 deg C until early stationary (~0.6-2E8 cells/ml). 2. Harvest in a 50ml sterile conical tube in GPR ce...