实验动物技术

Wholemount in situ hybridisationBased on Wilkinson protocol modified by Murray Hargrave (m.hargrave@cmcb.uq.edu.au) Koopman labA. SYNTHESIS OF PROBE1 Mix in the following order at room temperature:ste ...

BAC DNA has been purified successfully for microinjection by three principle methods: Anion Exchange Chromatography (Nucleobond Columns) CsCl Gradient or Sepharose 4B-CL Chromatography. Any of these m ...

This method was established in the University of Michigan Transgenic Core for our users. The method is used to purify BAC DNA for microinjection into fertilized mouse eggs and BAC transgenic mouse pro ...

Alkaline Lysis and CsCl Purification of BAC DNA for Transgenic Production.This method was contributed by Michelle Southard-Smith and Ron Chandler at Vanderbilt University Nashville Tennessee. This pro ...

Microinjection DNA PurificationWe have found the NucleoSpinTM Extract Kit (available from Clontech Catalog number K3051-1) is a simple and fast way to obtain microinjection quality DNA. This is not to ...

DNA ultrapurificationThe success of DNA purification can have profound consequences on the success or failure of any injection effort. This summary of techniques from several different sources was com ...

Isolation of DNA from Mouse Tail Biopsies1. Obtain tail biopsies from 2 to 3 week old mice: Hold mouse firmly at base of tail with one hand with the other cut off 0.5 to 1.0 cm of the tail tip with a ...

LacZ-cell Staining Materials: 1) 0.5% glutaraldehyde in PBS 2) 20mM K4Fe(CN)6o3H2O 0.44g into 50 ml PBS 3) 20mM K3Fe(CN)6 0.33g into 50 ml PBS 4) X-gal 2% in DMF 5) PBS containing 1mM MgCl2 6) Stainin ...

The purity of the transgene DNA used for microinjection is critical for the successful production of transgenic founder mice. DNA impurities in the form of bacterial endotoxins or organic contaminant ...

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 ...