Targeting Vector Submission for ES Cells
1. We will purify the DNA for you. Simply perform a restriction
digest on your cloning vector to liberate 50 ug of the transgene insert
from the cloning vector.
3. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to the Core lab and we will purify the DNA for microinjection from the digest. We use either the Gel Extraction Kit (Qiagen) or the Machery Nagel Nucleospin extract kit (Clontech) for purification of microinjection DNA. Please note, if you want use large DNA fragments such as bacterial artificial chromosomes, that there is a different protocol for the preparation of the BAC DNA for microinjection.
4. Bring a gel photo that shows you have a PCR assay for an endogenous mouse gene such as beta-globin. All DNA samples from potentially transgenic mice should give a positive result with an endogenous gene PCR. This will make sure that no transgenic founders are discarded because PCR inhibitors co-purified with the genomic DNA.
5. Bring a gel photo that shows your genotyping PCR. Test the PCR against 10, 1, 0.1 and 0.01 copy standards. The assay must detect the transgene at the single copy level, preferably 0.1 copies, when it is mixed with mouse tail DNA. Bring your calculations of copy number along with the gel photo. If you need tail DNA to set up the assay, we will give you some.
6. Bring a submission form with your contact information and billing information.
7. Drop off all your materials at the microinjection lab: Room 2526, Building MSRB I.
Transgene constructs are purified, quantitated, and microinjected into (C57BL/6 X SJL)F2 mouse eggs and surgically transferred to recipients. Other mouse eggs can be used for transgenic production when prior arrangements are made with the Core. Contact Thom Saunders regarding custom mouse strains.
Transgenic mouse production is a fee for service provided by the
Core. The Core prioritizes all requests for service on a "first-come,
basis. Your DNA will be added to the microinjection
queue in the order that it is received.
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