Matchmaker™Library Construction & Screening Kits User Manual PT3955-1 (PR742237) Published 20 April 2007 Clontech Laboratories, Inc. clontech.com Protocol No. PT3955-1 Version No. PR74 37 Matchmaker™ Library Construction & Screening Kits User Manual I. Introduction 4 II. List of Components 7 III. The ds cDNA was purified using chroma spin + TE 400 columns to select fragments above 400 bp, co-transformed with pGADT7-Rec into competent Y187 yeast cells using Yeast maker yeast transformation system 2 (Takara Clontech), and plated on -Leucine SD minimal agar media. 100 μl of a 1/100 dilution of the transformed cell suspension (15 ml) when plated on -Leucine SD Agar media with 72 h of Construction of MIC2 bait plasmids. (A) Schematic illustration of full-length MIC2, and its different domains used as baits in the yeast-two-hybrid screen.(B) Agarose gel electrophoresis analysis of different fragments of MIC2 amplified from T. gondii cDNA. M: DNA maker; 1: MIC2 ectodomain (aa74 - 596) that lacks propeptide and the sixth TSR motif; 2: A/I domain of MIC2 (aa74 - 270); 3: the Yeastmaker Yeast Transformation Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual (PT4087-1) System 2 (Box 1 of 2): - 2 x 1 ml Yeastmaker Yeast Transformation System 2 User Manual (PT1172-1) Yeastmaker Carrier DNA, denatured (10 mg/ml) - 20 µl pGADT7-Rec Vector Information (PT3530-5) pGBT9 (0.1 µg/µl; control plasmid) Lane M: DNA 5000 maker; USA) according to the user manual Following the protocols of the Yeastmaker TM Yeast Transformation System 2 kit (Clontech, Following the manufacture's protocols of the Yeast maker™ Yeast Transformation System 2 kit (Cat. No.630439, Clontech, USA), the transformants of pSTU2-APP plasmids were used as a positive control. The pPR3-N plasmid was used as a negative control. The pBT3-N-ORF047, pSTU2-APP, pPR3-N plasmids were transformed into NMY51, respectively. Following the manufacture's protocols of the Yeast maker™ Yeast Transformation System 2 kit (Cat. No.630439, Clontech, USA), the transformants of pSTU2-APP plasmids were used as a positive control. The pPR3-N plasmid was used as a negative control. The pBT3-N-ORF047, pSTU2-APP, pPR3-N plasmids were transformed into NMY51, respectively. MADS16 was combined with yeast library cell in 2× YPDA liquid medium using yeast mating method followed by incubation at 30˚C for 21-24 h according to the Matchmaker1 Gold Yeast Two-Hybrid System User Manual (Clontech, Cat. No.630489). The mating culture was plated on SD/-Leu/-Trp/-His agar plates for 7-14 d, and all the colonies were CHeflee Professional Dough Mixer, Smart Kneading and Fermenting Dough Maker, LED display &Adjustable Time, 230W 4.5 QT，304 Stainless Steel, White Dough Machine 4.0 out of 5 stars 42 $89.99 $ 89 . 99. Select dough cycle and let the bread machine do it's thing. Once the dough cycle is complete, roll out dough onto a floured surface and divide dough in 2. See notes below if only using 1 dough. 今天接着给大家翻译Takara Bio USA编著的《Matchmaker® Gold Yeast Two-Hybrid System User Manual》。 II. List of Components组分清单 Matchmaker Gold Yeast Two-Hybrid System (Cat. No. 630489) 将所有媒介物载体储存在-20°C，所有酵母 (酿酒酵母)菌株储存在-70°C，所有酵母介质袋储存在室温。 对于Yeastmaker酵母转化系统2，将载体DNA和对照质粒储存在-20°C，所有其他成分储存在室温。 Matchmaker Vectors • 50 µl pGBKT7 DNA-BD Cloning Vector (0.1 µg/µl) MAKER Transformation Syst