转基因实验

AP Buffer: 100 mM Tris-HCl pH 9.5 100 mM NaCl 10 mM MgCl2 PTM: PBS 0.1% Tween-20 2 mM MgCl2 Since this is generally done in conjunction with lacZ staining embryo/tissue is usually fixed as per la ...

NOTE: 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 microcentrifuge tube. 2. Add 500µl Tail Buffer a ...

These procedures were originally devised in Richard Palmiter's lab for use with tail dots (DNA spotted onto a nitrocellulose filter and probed for a transgene; quicker than doing southerns) and subseq ...

PCR screens must be designed to detect transgene DNA at the single copy level. Southern Blots analysis of transgenic mice need copy standards to estimate copy number. Copy standards are prepared by ...

This is how we test mice and rats for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with DNA genotyping and who need to establish a genotyping method. ...

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

1.Cut 1/2" - 3/4" of mouse tail. (Check with your institution's Animal Studies Committee for their recommendations as to how this procedure should be implemented). 2.Add tail fragment (or t ...

1. 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 scalpel or single edge razor blade. 2. A ...

This is how we test mice and rats for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with DNA genotyping and who need to establish a genotyping method. ...

1. Dissect embryos and place each yolk sac in a microfuge tube (can be frozen at -20°C prior to extraction). Original method cited for mouse toes or 2 mm tail Reagents: A) 25 mM NaOH / 0.2 mM ED ...

1. Remove 0.5 cm of tail into polypropylene microfuge tube (do not mince). (The tubes must have tight-fitting caps so that there are no leaks in steps 3 and 7 below.) 2. Add 0.5 ml DNA digestion buf ...

Need 1.5-2 x 107 cells from a 2 day culture. 1. Cells are harvested as normal washed x 1 in PBS then taken up at conc. of 1.2 x 10 7 cells/ml in cold PBS. 2. Take x2 Biorad curvettes A. 0.9 ml (107 ...

1. Cut the required amount of DNA (banded on a CsCl gradient) with the appropriate restriction enzyme and check for complete digestion by running 500 ng on a minigel. The DNA concentration should be n ...

Mice are vasectomized to be mated to females used for embryo transfer. The female becomes pseudopregnant her body geared for a pregnancy because she has been mated. Vasectomy is performed on mice at 6 ...

Mice are vasectomized to be mated to females used for embryo transfer. The female becomes pseudopregnant her body geared for a pregnancy because she has been mated. Vasectomy is performed on mice at ...

Bleed mice: Obtain 300-400µl from mice of various ages (older mice that have been in the room for an extended period) and strains (include nude mice if in the room). 5-8 mice is a good number. ...

Injection and Holding Pipettes The glass capillary tubing used should be thin walled borosilicate glass without a fibre. e.g. Clark Electromedical Instruments ...

These protocols have come from the transgenic mouse list and are intended as a guide none of them have been tried in this lab Cell(s) transferred in small volume of water to a .5 ml tube and lysed by ...

This method is modified from Dr. Lyn Corcoran (WEHI)

This technique appeared as an article in Biotechniques Vol.14 No.3 pp412-414. This article is a report by Kristian Gundersen Theresa Hanley and John Merlie on the use of micropipette bevelling to incr ...