protocol-PCR genotyping - (Apr/25/2008 )

1. Can any body suggest a protocol to isolate DNA from mouse tail or any good kit for PCR genotyping?

------Are these PCR's any different than regular PCRs?

2. Can the plasmid DNA of the same transgene be used as a positive control to tell the exact band size?

Thanks for your help.
jhilmil

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hey!

phenol chloroform extraction works fine, but you can try just to precipitate dna with isopropanol as well. depends on how good your pcr works, just give it a try under different conditions. it may save you a lot of work later.
as a positive control you can use dna from a mouse that has been identified positive in southern blot.

squishy

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Thanks for sharing ur protocol,

We dont have any positive transgenic mouse ( its our first Tg mouse), so can I use my plasmid DNA containing cDNA as a positive control ?

What concentartion of genmic DNA shold be used in positive/negative control?

Thanks for ur help.

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1. Yes you can use plasmid DNA as positive control, as long as the same area does not exist in the wt mouse genome.
2. Beware of contamination.
3. Use plasmid in picogram range. Preferably, have two +control tubes, in one mix plasmid with your tail genomic DNA, in another plasmid alone.
4. Use genomic DNA about 100-500 ng/reaction. It depends upon the copy number of integration.
5. Look up the following protocols:
http://search.vadlo.com/b/q?sn=158621799&a...l+dna&rel=0
http://search.vadlo.com/b/q?sn=158621799&a...yping&rel=0
Don't be confused:-), choose one and use the rest as guide.

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There are some dna extraction kits for tissue, they are very easy to use and less hazardous than phenol.

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Yep, I would definitely get out of phenol business!

In fact, if you look at a lot of these protocols, for ordinary genotyping PCR, you don't even have to purify DNA- just lyse the tails and use 1 ul of lysate for PCR.

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Thanks to all of you for the information, "cellcounter" the links were very useful..

I have got a beautiful gel after PCR, now i am ready to make crosses between mouse which showed a right size band, my question is how to make crosses between positive founder pups for establishing the transgenic mouse colony?

I have positive 1-male and 4 females out of 23 pups. I had marked the ears so I can put

a) cage1- positive male with 2 non-transgenic females, once females are pregnant, i can seperate those females
cage 2-5: positive 4 females with one non-transgenic male ecah in 4 cages

1) Is my thinking correct?

2) can I use the non-transgenic pups seen in my PCR genotyping for mating ?

3) how to check that the mating is done and a female is pregnant?

4) Can I mate positive male with a positive female at this point ?

Thanks for the help !

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1. It is good to segregate transgenic alleles to the point that each subsequent generation gets the same gene dosage. So, I generally mate the founders with wt Bl6, and (sometimes other inbred backgrounds too), and after a few generations, depending upon your patience, you can start breeding hemizygous.

2. I would not use non-transgenic pups. Just use pure inbred mice from Jackson or Charles river.

3. Female gets plugged, which you can check the next morning, but in this case, just leave them alone for a few weeks!

2. You know what, this entire matter requires a lot of considerations, that I can not write them all here. So, I would suggest that you read some about breeding up a transgenic colony and then ask some practical questions that are not covered.
http://search.vadlo.com/b/q?sn=158621799&a...ding+&rel=0

3. You may also want to check out some OXford/Humana press books that I am sure should be available in your library. There are just too many things involved here for me to write-up.

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