Co Precipitant - Glycoblue Ambion? - (Jun/13/2005 )
I was considering using a co-precipitant when isolating small quantities of RNA and DNA. My concern is whether or not the co-precipitant will have any effect on downstream reactions such as qRT PCR or Methylation study. Also will it have an effect on quantification using a spectrophotometer? Could anyone explain how exactly a co-precipitant interacts with a nucleic acid molecule?
Specifically I was looking into Ambions Glycoblue in conjunction with Qiagens RNA/DNA mini kit. Has anyone had any experience with this?
I don't have any experience with glygoblue... I can tell you it is glycogen with an inert blue dye attached to it to make the pellet more visible upon precipitation.
It works by 'surrounding' the dna/rna and since it is a long chain proteoglycan it is easy to precipitate, taking the rna/dna with it. It actually works quite well.
I have not used ppt dna or rna on real time pcr to methylation studies... so I cannot difinitively speak to that, but it has not interfered with downstream applications I have used. It does not interfere with A260 readings.
I have used PelletPaint for downstream applications such as PCR with no problem at all. There is also pellet paint NF for fluorescent sequencing applications. Check the product sheet that comes with Glycoblue it should tell you what downstream applications the DNA/RNA can be used for. Generally speaking these co-precipitants should be inert and not affect downstram apps, otherwise they won't be too useful!
Does it affect downstream cloning procedure? I usually have no problem in cloning. But this time, I used the glycoblue for the first time, just to make it easier to precipitate my vector. Everything looked fine, until I isolate the plasmids from the colonies that I got. I found most of the reombinant plasmid are even smaller than their parent vector.
Can anyone tell me whether it is due to glycoblue interfering?