Purify ChIP DNA using Qiagen spin filter - (Nov/30/2009 )
Hello,
I am using Milipore CHIP kit, The problem is i am running out of these kits very fast and the price tag is really high.I am thinking to try Farham lab protocol.
For now, i noticed i have enough reagents from last 2 millipore kits to do 10 samples but i have only 2 spin filter columns left. and i cant buy the columns seperately from millipore.
Keeping in mind that these are for DNA purification, i am thinking to use Qiagen DNA purification column.
Have anyone tried to use different column with millipore CHIP kit?
Can i do this?
thanks,
Yes you can use Qiagen PCR purification kit for the final DNA purification, but you will need additional PB buffer because in the first step you need to add 5x PB buffer to the DNA which has a volume much bigger than that for PCR reactions. You can buy separate bottles of PB buffer from Qiagen.
The Qiaquick kit from Qiagen works just fine with the millipore kit. I make use of the Millipore magna-chip kit as well and have run into the same problem. I wrote out all the ingredients that come with the kit and how many I use per ChIP sample (I'm dissecting out brain tissue, so I use different amounts than Millipore recommends) and which reagents are my limiting reagents. For me it was the Cell Lysis Buffer and the Protease Inhibitor cocktail.
Like you, I tried to order reagents individually, but they don't sell them; but if you call Millipore they will give you the composition of many of their reagents (e.g, now I make my own cell lysis buffer) or buy them from elsewhere (I've been using the Halt protease/phosphatase inhibitor cocktail from Pierce; has worked well so far). I think it's quite obnoxious that they don't sell components individually given that various labs have different reagent requirements....
MM
I have a similar question. I used Sigma-Aldrich Imprint CHIP kit, but those DNA isolation columns in kit were awfull, hard to open, uneven size (some of them didn't fit in the centrifuge), crappy plastics and what's more important the elution volumes were different and about only 2/3 of the volume I put in. Couldn't believe that.
If I need to repeat it, I would rather use other columns. We have Qiagen MinElute and Qiaquick columns (various kits, Gel Extraction, PCR purification maybe even reaction cleanup) and DNeasy Blood and Tissue colums. Which do you think would be most suitable for CHIP DNA purification? I'm mostly worried about buffer compatibility (CHIP lysate with column binding conditions) in that case.
Trof on Fri Jul 20 14:17:51 2012 said:
I have a similar question. I used Sigma-Aldrich Imprint CHIP kit, but those DNA isolation columns in kit were awfull, hard to open, uneven size (some of them didn't fit in the centrifuge), crappy plastics and what's more important the elution volumes were different and about only 2/3 of the volume I put in. Couldn't believe that.
If I need to repeat it, I would rather use other columns. We have Qiagen MinElute and Qiaquick columns (various kits, Gel Extraction, PCR purification maybe even reaction cleanup) and DNeasy Blood and Tissue colums. Which do you think would be most suitable for CHIP DNA purification? I'm mostly worried about buffer compatibility (CHIP lysate with column binding conditions) in that case.
I would think, given the small yield in a ChIP assay, that the MinElute kit would be most appropriate.
Mighty Mouse on Wed Dec 9 02:35:44 2009 said:
The Qiaquick kit from Qiagen works just fine with the millipore kit. I make use of the Millipore magna-chip kit as well and have run into the same problem. I wrote out all the ingredients that come with the kit and how many I use per ChIP sample (I'm dissecting out brain tissue, so I use different amounts than Millipore recommends) and which reagents are my limiting reagents. For me it was the Cell Lysis Buffer and the Protease Inhibitor cocktail.
Like you, I tried to order reagents individually, but they don't sell them; but if you call Millipore they will give you the composition of many of their reagents (e.g, now I make my own cell lysis buffer) or buy them from elsewhere (I've been using the Halt protease/phosphatase inhibitor cocktail from Pierce; has worked well so far). I think it's quite obnoxious that they don't sell components individually given that various labs have different reagent requirements....
MM
I have the same problem as you! I also used all cell lysis buffer and my kit is from Millipore. They say it for 22 assays. I think it's just for one sample with small amount. I have several samples every time so it's far not enough for me. I found there are many different cell lysis buffer and nuclear lysis buffer in web. But I do not know which one can work with Millipore Kit. I really appreciate if you can give me suggestion about making my own cell lysis buffer.
our lab is using two different columns from Enzymax.net, which work very well and price is very good as well. I like the micro spin column, you can elute DNA in just 5ul. The other column is the mini spin column and the elution volumn is 40ul. I think the design for micro spin column is different, which has no buffer retension, maybe that is why you can get 5ul elution volume.....
Dear all,
we now used Qiaquick kit to purify our DNA after the Chip and it work very well. Just remember that it can be a problem if you want to quantify the eluted DNA.
I have been using the Qiagen DNAeasy tissue kit to extract the DNA after ChIP assay and it worked fine. Similarly, I used the Macherey-Nagel DNA purification columns