gDNA extraction from mouse ear for PCR - "Hot Shot" extraction (Sep/29/2008 )
I need some help,
This is the thing:
I have transgenic mice and need to test every new born to see if it has the gene of interest. The way to do so is by PCR, as I only receive a piece of ear I looked for genomic DNA extraction protocols; I've been using one in which I leave the sample ON with a buffer and proteinase and then, take the supernatant and do alcohol precipitation. The problem with this is that sometimes it works just fine (since when doing PCR I get the control band -actin- and if it's TG I get the band of interest), while others it's not so efficient........sometimes controls show no band, sometimes I get no band for TG but it turns out to BE a transgenic after all!!!!!
Anyway, I need other strategy..
I found this "Hot Shot" protocol for ears exclusively (the other was for tails).
15' at 95ºC with NaOH 20mM
then TRIS 40mM
and that's it. you use the supernatant (more or less 150ul starting with a 2x2mm ear piece), for PCR.
actin controls are OK, maybe reaction is "dirty" as when you see the gel there is something behind the band.
I've already done dilutions and works fine even with 1:100, and precipitation of DNA before use...both things improved the results, giving more "clear" bands...............this is for actin though I still don't have TG bands in any case!!!!!!!
I' don't know what else to do!! It has no sense the only thing.......is primers maybe??could it be??? it's the only thing different from the controls!
ONE IMPORTANT THING POSITIVE CONTROL (which is gDNA extracted once with the other strategy) IS OK....YOU GET A BAND WITH THIS ONE!!!!!
So what can be happening??? any ideas??
As we are talking about PCR, how can I treat the amplicon to use it as template for the next PCR reaction (speaking about the positive control, to not run out of it)
hope you can help!
how clean is your DNA???maybe your transfering some debris of the DNA extraction that coul interfere with the PCR reaction. Maybe a salting out protocol is better option. Add DMSO, nonidet p40 to the PCR reaction.