PCR product precipitation - (Oct/17/2005 )
Hi,everyone here.I am a new in this forum.
Recently I am working with PCR-DGGE method to study environmental microoganism diversity.
I use 50 ul volume in PCR, this volume seems a little larger for DGGE.
so I have to reduce my products volume before loading.
The method I use is as follows:
add 5 ul 3M NaAC and 40 ul isopropyl alcohol to 50 ul product,and put them in -20C for at least 2h,then centrifuge(1o,ooog, 15min),wash the precipitate with 70% cold ethanol,centrifuge again(10,000,5min).Dissolve the precipitate in 20 ul TE buffer.
But the problem is the pricipate is hard to dissolve in TE again.I have tried this 2 times,but the result was same .
I hope someone can help me.Thanks
Precipitation with isopropanol frequently produces pellets that are somewhat difficult to dissolve. A few suggestions:
1. Try ethanol precipitation. Use 1/10 volume NaAc and 2 volumes of 100% ethanol.
2. Resuspend in 10 mM Tris, pH 8.5. Use of an alkaline pH will help the DNA dissolve. I'd also leave out the EDTA, especially if you're using the DNA for PCR -- the EDTA will chelate the magnesium.
3. As an alternative to precipitation, use a PCR cleanup kits (I use Qiagen's kit).
Thank you for your advice.
Another question,I have never used PCR cleanup kit before.What I want to know is that can I use the kit to reduce the volume to what I want? Thanks
do you want to use the column after your precipitation? isn't possible to use it directly?
for resuspension, heating helps great to dissolve pellet (50 to 65° for 10')
If precipitation method is unuseful,I will try the kit.