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#1
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| I'm looking for a DNA size standard I can use with southern blots. I don't want to deal with radioactive gel apparatus, so I don't want to end label my ladder before I run the gel. The ideal solution would be something dyed so I could see with the naked eye on the membrane, and mark the sizes with radioactive ink. Is there something like this available? Here's an idea, I can see the DNA ladder in my gel under UV. Could I inject a small amount of P32 into each size standard, and then transfer so the size standards would be visible on the membrane? I've seen protocols for making size standards from lambda DNA, but these require probing with lambda DNA. If I do this, would I include the lambda probe in the hybridization with my probe? Do I need to reprobe with the lambda probe? |
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#2
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| On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote: Just stain the membrane with ethidium Br or other fluorescent dye. After the transfer you usually can see your ladder quite well. In Southern (depending on the complexity) it is difficult to avoid seeing the ladder due to non-specific hybridization . my2c |
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#3
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| On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote: Just stain the membrane with ethidium Br or other fluorescent dye. After the transfer you usually can see your ladder quite well. In Southern (depending on the complexity) it is difficult to avoid seeing the ladder due to non-specific hybridization . my2c |
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#4
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| On Jun 25, 3:38 pm, Ed Siefker <[Only registered users see links. ]> wrote: Just stain the membrane with ethidium Br or other fluorescent dye. After the transfer you usually can see your ladder quite well. In Southern (depending on the complexity) it is difficult to avoid seeing the ladder due to non-specific hybridization . my2c |
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#5
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| "Ed Siefker" <[Only registered users see links. ]> wrote in message news:[Only registered users see links. ].n et... I tend to simply add a *minute* amount of the ladder to my probe during the labelling reaction. Then you see the ladder as well as your probe... but of course, you have to be sure there won't be crosshybridisation. Jose |
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#6
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| Ed Siefker schrieb: In our lab, we have frequently observed cross-hybridisation of labelled probes with the DNA Ruler Ladder Mix from Fermentas (no affiliation). Interestingly, this happens only with this marker, but not with others like Lamba-Pst - so you could give it a try if this wouks with your probes, too. We also usually photograph our gels with a ruler next to it, so by measuring the distance from the wels to our band, we can interpolate the sizes. Good luck Kay |
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| size , southern , standards |
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