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PROTOCOLS:


DNA Preparation for Microinjection or Electroporation

This protocol was developed by Jan Parker-Thornburg at The Ohio State Universtiy


1)Run the DNA digest on an agarose gel:

2) Purifiying the DNA fragment.
You can dialyze overnight against 2 liters of injection buffer at this point, but it's really not necessary.

This method of prepping fragments is proving to be a really reliable and clean method. I have used it on pieces as large as 18 kB (that't the reason I went to spin columns in the first place). The spin columns are simply a GeneClean kit with a filter to catch any residual glass beads. The literature that they send says that the columns are good for DNA from .2-200kB.

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Pronuclear Injections:

Egg Production for injections
To obtain a large quantitiy (>250) of eggs for injection, sexually immature FVB/N females (4-5 weeks of age) are superovulated by using consecutive PMS and HCG hormone injections. Females are mated to stud males immediately following the HCG injection.

Harvesting the eggs
Eggs are harvested the next day from the ampulla of the oviduct of the mated females. Eggs are treated with hyaluronidase to remove nurse cells, and are then washed through several dishes of M2 media. Fertilized eggs are then stored in M16 media at 37oC and in 5% CO2 until injection.

Injecting the eggs
30-50 eggs are removed from the incubator at a time for injection. Each egg is individually injected with the DNA fragment of the day under high magnification. When each egg in that group has been injected, all the eggs are returned to the incubator. This procedure is repeated until all eggs have been injected. At the end of the injection period, eggs which have not survived injection are removed from each group.

Implanting the eggs
Injected eggs are then implanted in groups of 10-15 bilaterally into the oviduct of pseudopregnant females (females which have been mated to vasectomized males). The animals are allowed to recover from anaesthesia on a warming plate, and then returned to the animal room. They are kept under sterile conditions throughout their pregnancy.

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Morula Aggregation:
This technique has been beautifully described by
Andras Nagy (one of the developers of the technique) in his web page. Click on his name to go there.

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PCR for Determination of Transgenic Mice

This protocol was developed by Jon Neumann at the University of Cincinnati

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Tail DNA Preps for Genomic Southerns

This protocol was developed by Arun Subramaniam from Proctor and Gamble

Digestion of the tail clip:


Restriction digests of genomic DNA:

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Obtaining a University Approved Animal Protocol
Prior to performing any transgenic work, we require the investigator to have an IACUC approved animal protocol. Information regarding the procedures for obtaining the protocol can be accessed using the
ULAR web page--just click on the highlighted ULAR.

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Mating Protocol

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