实验动物技术

Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a CarrierKatherine KolorReferences: Schiestl and Gietz (1989) Curr. Genetics 16:339-346 Gietz et al (1995) Yeast 11:355-360 ...

DNA isolation from yeast 1) Start a 5 ml o/n culture 2) Pellet o/n culture in a purple screw cap tube (this may take several spins) and dump s/n.3) Add 100 µl of STET to pellet and vortex briefly.4) A ...

Genomic DNA Miniprepgrow 5 ml cells over night at 30 °C spin wash once with 1 ml H2O resuspend in 500 µl lysis buffer add acid washed glass beads to 1.25 ml vortex 2 min recover liquid phase with blue ...

Joel Huberman's DNA isolation procedure with modifications by Bonny Brewer1. Kill cells at mid log phase by adding Na Azide to 0.1%. Immediately chill culture by adding it to frozen 0.2 M Na EDTA. (4 ...

Sucrose Gradient Protocolfrom Song et al. 1996 Modified by Ginger HoffmanGrow a 2 ml culture 24 hr. at 30oC in selective media When culture is ready use it to inoculate about 55 ml (50 ml plus 5 for ...

Whole Cell ExtractsREFERENCE: Hoffman G. Garrison T. R. and Dohlman H. G. Analysis of RGS proteins in Saccharomyces cerevisiae Methods Enzymol.344:617-631 2002. -Use sterile technique and sterile so ...

Yeast RNA Miniprepgrow cells to mid-log phase (10-20 ml) (or take samples from time courses 5-25 ml) spin cells discard supernatant wash cells once with 1 ml cold RNA buffer freeze pellet in dry ice ( ...

Yeast mini whole cell extract using glass beads for western analysisSteve HahnLast modified Fri Oct 16 1998Before trying this method try rapid protein prep which involves boiling yeast in SDS PAGE buf ...

ß - GAL Filter Assaystreak yeasts to single colony replica plate onto NC-filter (leave 10 sec.) use tweezers to lift filter and slowly submerge in liquid Nitrogen for 1 min place on back side of a pet ...

Making library DNA from the DNA we send youThe library is distributed as 18 individual pools in the form of DNA. You will be sent about a microgram of each. Transform a suitable amount into E. coli ( ...

Replication timing by comparative hybridizationM. K. RaghuramanReference: Friedman K. L. M. K. Raghuraman W. L. Fangman and B. J. Brewer (1995) Analysis of the temporal program of replication initiati ...

Replication timing by density transfer M. K. Raghuraman 1. Grow cells at least 7 generations in0.1% (w/v) C-acetate or glucose 0.01% (w/v) N-ammonium sulfate Amino acids/supplements (as required added ...

Vectorette PCR of Yeast DNACarl Friddle1) Cut 1-3 µg of clean DNA overnight with 8-10U of blunt cutting enzyme in 20µl Most problems come from dirty uncut DNA. Phenol/glass bead/RNase prepared DNA wor ...

　1 甲醇酵母表达系统的特点　　1.1 宿主　　七十年代巴斯德毕赤酵母曾被用于生产单细胞蛋白（SCP），有很好的发酵基础，菌体密度可达100g/L干重。其生长培养液的组分包括无机盐、微量元素、生物素、氮源和碳源，廉价而无毒。它能在以甲醇为唯一碳源的培养基中快速生长，其中醇氧化酶AOX――甲醇代谢途径的关键酶可达细胞可溶性蛋白的30％。而在葡萄糖、甘油或乙醇作为碳源的培养细胞中则检测不到AOX。AO ...

问：如果诱饵蛋白对酵母细胞是有毒的，该怎么办？答：在有些情况下，在液体培养基中培养不好的菌珠可以在固体培养基上生长得很好。重悬克隆于1ml的SD/�Trp，接着将重悬液平铺于5 个100-mm的SD/�Trp平板。在30℃下温浴，直至平板上的克隆互相粘在一起。用5ml 0.5X YPDA刮下每块板上的克隆，并收集到一管中。接着就使用这个细胞重悬液进行正常的杂交反应。问：我的诱饵蛋白能直接激活报告基 ...

1. Introduction and BackgroundThere is a great need for general methods to characterize the proteins that contemporary biology makes available. The list of such proteins needing further characterizati ...

DNA preparation from mouse tails General precautions to be followed during handling of any genomic DNA sample: Avoid Shearing of the DNA by limiting mixing to inversion and gentle shaking. Vortexing o ...

Blastocyst embryo transfer into pseudopregnant recipient female mouseBlastocyst transfer is best performed after allowing injected embryos a little recovery time in culture. This allows better evaluat ...

ES / MEF cell culture and electroporation of targeting constructDay 0 One frozen vial of Murine Embryonic Fibroblasts (MEFs) is thawed quickly in a 37oC water bath. When the last bit of ice is melted ...

Always fill-in a form for each mouse you find/submit and make as many notes as possible about his behaviour health status or gross features. Always weigh the mouse. if mouse is found dead : Almost inv ...