实验动物模型

Mouse Tail (or organ) Biopsy DNA ExtractionLysis Buffer Proteinase K25 ml 1M Tris pH 8.0 (FC 50mM) 100 mg dry Proteinase K (Sigma #2308)50 ml 0.25M EDTA (FC 25 mM) 4.7 ...

1. Obtain the last 2mm of tail and place directly into 200ul 1X PCR Bufferwith Nonionic Detergents (PBND) in a 1.5ml microfuge tube. (Tails can bestored at frozen in PBS or PBND until use.)2. Add 1.2 ...

Sepharose 4B-CL Chromatography BAC DNA Purification for Transgenic ProductionThis method was contributed by Wes Dunnick at the University of Michigan Medical School in Ann Arbor Michigan. The approac ...

Purification of Gene Targeting Vector DNA for Electroporation1. Purify plasmid from bacteria.We recommend the Qiagen EndoFree Plasmid Maxi kit for the purification of the targeting vector plasmid from ...

Sample Preparation for Microinjection General CommentsMicroinjection is the loading or transfer of a dissolved substance into a living cell. The microscopic tip of the glass microcapillary has an inne ...

Use 4ml polycarbonate snap-cap tubes for all steps. Rocking of tubes should be performed at all times. All steps are carried out at room temperature unless otherwise stated.Day 01. Dissect embryos in ...

X-gal staining of embryosProtocol supplied by S. P. Yee (spyee@julian.uwo.ca) and Joe Chan (cchan@hgmp.mrc.ac.uk).ProcedureDissect embryos in PBS Immediately transfer the embryos to fixative at 4oC i ...

Preparation of Copy Standards for PCR GenotypingPCR screens must be designed to detect transgene DNA at the single copy level. To demonstrate this level of sensitivity non-transgenic tail DNA is spike ...

Preparation of Mouse Tail DNA for Dot Blots or PCRThese procedures were originally devised in Richard Palmiter's lab for use with tail dots (DNA spotted onto a nitrocellulose filter and probed for a t ...

Tail Preps: DNA Isolation From Mouse Tails Without PhenolNOTE: THIS IS FINE FOR SOUTHERNS BUT NOT FOR SCREENING BY PCR. (from Ruixia 7/99 from protocol by Stef Oehen 7/94). 1. Put 1 cm tail in 1.5 ml ...

DETECTION OF ß-GALACTOSIDASEThe E. coli lacZ gene encoding ß-galactosidase (ß-gal) is the classical histochemical reporter gene (Beckwith 1980). It can be detected using a variety of substrates all o ...

RNA-isolation (TRIZOL method)Isolation of RNA from whole worms using TRIZOL reagent (Gibco-BRL).1) Wash worms from a 60 mm plate with 1 ml DEPC-treated water.2) Spin down worms at 4000xg in microfuge ...

Nematode Genomic DNA IsolationMaterials:Rich Agarose Plates: 50 mM NaCl 0.75 % BactoPeptone 5 ug/ml cholesteroundefined 1 mM CaClundefined 1 mM MgSOundefined 25 mM K-PO4 (pH 6.0~undefined 1.5 % agarose (not agar) * add after auto ...

Cleaning Worm Stocksby Michael Koelle4/6/94There are two kinds of contaminants on worm plates:1. Fungi: these contaminants can come from the plates or bacteria so it is best to leave plates out after ...

Overview Using this procedure it is possible to follow the development of pollen mother cells through to mature pollen. Sterile males were made using irradiation mutagenesis of Landsberg erecta seed ...

Simplified Arabidopsis Transformation Protocol(Brief version for those who are familiar with the method)Steve Clough and Andrew Bent University of Illinois at Urbana-Champaign. Our present protocol (C ...

Culturing Wormsby Michael Koelle1. Bacterial strain for feeding worms: OP50 a uracil auxotroph. Streak OP50 out on a 9 cm NGM agar plate grow overnight at 37°. Can then parafilm the plate and keep it ...

Procedure1. Immerse Arabidopsis seeds in 10% Household Bleach for 20 min. 2. Rinse the seeds twice with 500 ml of sterile ddH2O and allow them to dry overnight in a laminar flow hood. 3. Push the res ...

Green lab protocol for vacuum infiltration transformation of Arabidopsis Step by step picture demonstration This protocol is adapted from protocols by Nicole Bechtold (Bechtold et al ...

Development of techniques for primary culture of C. elegans embryonic neurons Laird Bloom MIT from Ph.D. thesis Massachusetts Institute of Technology 1993 Introduction One of the major limitations of ...