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Genotyping - Primers for PCR genotyping (Sep/20/2007 )

I need to design primers to do PCR genotyping for my transgenic mouse.

Which region will be best to make sure that its expressing the transgene?
Should it cover both the promoter and the gene region or just gene?
Is it prefferable/neccessary to PCR the whole gene sequence?

Since i will get to start genotyping after few weeks, is there any way i can standradize the PCR conditions? In another words, can i use any mouse gDNA to optimize the PCR conditions with my primers?

Why it is preferable to do RT-PCR, when we can check the expression of the transgene by PCR using gDNA of a transgenic mouse?

Thanks for your help/suggestions.

-jhilmil-

design primers so that you can amplify the junction where the transgene is inserted. You need to standardize your protocol. Try to amplify using a sample gDNA from some mouse.

-scolix-

Hi,
scolix is right about checking the junctions. however u can also try

1. design the primers for ur promoter end region and gene mid region. (ensures that ur gene with promoter is there)
2. design primers for ur promoter as well as ur genes (further ensures that the gene and promoter are integrated intact in the transgenics)
3. u need to do this apart from RT as this ensures that gene is there (just in case its not expressing, as might happen in some case) and treat the subjects (plants/animals) as transgenics for handling separately
4. later u might have to do chromosome walk to see the correct site of integration of ur gene.

hope i was of some help....!

-GeneDoc-

QUOTE (GeneDoc @ Nov 30 2007, 06:37 AM)
Hi,
scolix is right about checking the junctions. however u can also try

1. design the primers for ur promoter end region and gene mid region. (ensures that ur gene with promoter is there)
2. design primers for ur promoter as well as ur genes (further ensures that the gene and promoter are integrated intact in the transgenics)
3. u need to do this apart from RT as this ensures that gene is there (just in case its not expressing, as might happen in some case) and treat the subjects (plants/animals) as transgenics for handling separately
4. later u might have to do chromosome walk to see the correct site of integration of ur gene.

hope i was of some help....!


Thanks for your suggestion.

How to do chromosome walk to see the correct site of integration of my gene?
How do you determine copy number and number of integration sites?
Thanks!

-jhilmil-

copy number is probably easiest by southern blot - you will need a restriction enzyme that can cut your DNA once in the insert so you can see if you have inserts concatemerised (i.e. one after the other at the same insertion site).

-bob1-

QUOTE (bob1 @ Apr 17 2008, 09:48 PM)
copy number is probably easiest by southern blot - you will need a restriction enzyme that can cut your DNA once in the insert so you can see if you have inserts concatemerised (i.e. one after the other at the same insertion site).



So, I need to use genomic DNA isolated from tail piece for southern blot, am I correct?
Thanks!

-jhilmil-