southern and restriction enzyme - what enzyme to choose (Mar/30/2006 )

hi I plan to to southern blto on my tail dna extraction to see if my mice are transgenic.

how do I know wich enzyme to choose.
any exemple?
should the enzyme be not in my transgene?

thanks

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I am not an expert, but I would think a good enzyme could still be in your transgene. The main consideration is to know ahead of time what bands you will see if it worked, right?...so maybe don't use an RE that spans your probe

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hi

i am making at the moment also a southern analysis to determine the number of copies in my transgenic poplar lines. what REs you need is dependent on your construct insert in your lines.
first you have to choose such enzymes which dont cut in the sequenz of your gene probe by hybridising, enzymes should cut only one time in your construct, near by left and maybe right side of your sequenz. one time cutting is important because if you have two cutting sites you will get always the same bandle lenght on your film later. by one cutting site you have always different lenght to your construct sight because the enzyme will cut in different distances on your genomic dna outside your construct sequenz.

take two or three different enzymes best the common enzymes like ECORI,BAMHI,XBAI,XHOI,HINDIII but maybe not PSTI and SALI,in my digstiones it never works fine with these two ones,

good luck

jost

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[hi
thanks for the adv ice.
I have another question.
Ihave a collegue who generate transgenic mice. but in his construct thee is no polyA signal.
is it a problem then for detection the transgene by PCR, RTPCR and southern.

thanks

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I think perhaps the only thing different there, is that when he generates cDNA for RT-PCR he needs to use either specific primers or random primers, but not oligo(dT) primers

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hi
well the thing is that Igot a PCR product, I got RTPCR product, I got something on the western blot.
so now Iwant to do southern blot. I did it in not radioactif way. Ididn't get a band.
so I was wondering since the construct doesn't have polyA, maybe that's why I don't see band.
but it shouldn't be a problem because the polyA is the RNA step, and for southern I look at the DNA level.
right?

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I'm not sure why the polyA would matter...I do not think that would have anything to do with your Southern blot, I think you are right.

have you done a positive control?

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