BSP to test imprinted genes DNA methylation of oocytes - no specific or smear bands! (Nov/17/2007 )
Hi, all
I have a big headache when testing imprinted genes DNA methylation of oocytes. Around 50 MII oocytes are embeded in a small low melting point (LMP)argarose and modified using traditional bisulfite coversion. This method work well in our lab even when treated only one embryo in the bead and then used the one bead as the template directly. But I fail to get specific bands insteads of nothing or smear ones by the same way treated oocytes gDNA. My procedures are following.
1.collect about 50 MII oocytes in a pcr tube and add 2ul 20mg/ml protein K, put the tube in 37 ℃ for 30-60 min.
2.add 1.5ul 3 M NaOH and put the tube in 37 ℃ for 15 min
3.add 8ul 2% argarose to embed the DNA in the bead
4. 50 ℃ overnight(14-16h) in 3.8 M bisulfite
5, wash the bead with TE 3X15 min, 3M NaON 2X15 min and again TE 2X15 min,finally H2O.
After washing, use the bead directly or store them at -20℃.
I don't obtain specific fragments after nest-pcr. I don't know why and I come here for helps.
Any advices are welcome. Thanks in advanced!
xingweil
Are you using primers that have already worked in your lab? I don't see a obvious flaw in your protocol but I do not do homebrew myself...
Yes, the primers work well in the lab.
Several days ago, I found an quite interesting and confused phenomenon. I did the following experiment and the results made
me surprised. I cann't amplify the SNRPN using gDNA from oocytes, although I have confirmed the template is 0k cause I have
successfully amplified H19 used the same template. I really don't know why? Is it SNRPN more difficult to ampliy and my
template don't meet both quality and quantity.
gDNA from oocyte gDNA from liver
H19 ok ok
SNRPN nothing ok
Welcome for interpretation!
xingweil
xingweil,
just wondering, where is your desulphonation step in your conversion protocol? after the second denaturation we add ammonium acetate to the DNA (we don't do beads) to remove the sulphite adduct on the DNA and then precipitate.
As for your SNRPN amplification failing in oocytes and not liver that is a strange result. I would say maybe it's GC-rich but you are able to get an amplicon in liver. that is perplexing.
Nick
Nick,
Thanks so much.
It seams no desulphonation step in my modification treatment. After the wash steps with TE, NaOH and water, I use the beads as template directly and without doing desulphonation and precipitate. I embeded around 50 mouse oocytes in a bead although someone reported that only 5-10 oocytes are enough. I never try to recover the converted gDNA embeded in beads cause I think the amout of gDNA is so small and if more steps made, more gDNA lost. I will try to use gDNA extract kit and modification kit to overcome those problems.
I am not sure if the stochastic characteristic of PCR or something else resulted in my perplexing result. After all, I didn't do the repeat experiment.
Xingweil
just wondering, where is your desulphonation step in your conversion protocol? after the second denaturation we add ammonium acetate to the DNA (we don't do beads) to remove the sulphite adduct on the DNA and then precipitate.
As for your SNRPN amplification failing in oocytes and not liver that is a strange result. I would say maybe it's GC-rich but you are able to get an amplicon in liver. that is perplexing.
Nick