We are having troubles with the isolation of free circulating DNA. We already tried QIAamp free circulating kit (Qiagen). Can you recommend other protocols?
Saliva is so easy!!!!!! But I don´t used this kit!!!!! I have another method!!!!
are you using the human sera for your sample? maybe you can use the promega KIT, and may this link will be help you
http://www.promega.com/~/media/files/promega%20worldwide/north%20america/promega%20us/aacr/a%20novel%20method%20for%20efficient%20and%20hands-free%20purification%20of%20circulating%20dna.pdf
one option:
1)perform a protein precipitation, spin and collect supernate
2)precipitate DNA using 100% ethanol and immobilize on a standard extraction column.
3)wash with AW1 followed by AW2
4)elute DNA w molec. H2O
-all the reagents are standard Qiagen.
ps-is there some reason you have not tried the standard protocol starting w protease K, lysis buffer, etc?
We use the QIAamp DSP Virus Kits as routine for our diagnostics work with cell free foetal DNA. Although, all our extractions are done on the QIASymphony.
Thank you for your help!
But we already tried the standard QIAamp circulating nucleic acid kit and didn't get sufficient concentrations.
Hi Sandra,
As it was said, use higher volumes of plasma. In addition, this years has been released a new version of the kit from QIAGEN that works perfectly.
QIAamp free circulating kit (Qiagen) is perfect. I have very good experience with this. You may be missing some crucial step.
I want to add more information to my previous answer to this question. I will suggest using Qiagen's QIAamp Circulating Nucleic Acid Kit (Cat# 55114). I get very pure preparation of DNA which I have used for droplet digital PCR and NGS. I personally have compared extraction from 10 plasma sample using Qiagen and Zymo kits and the recovery by Zymo kit was almost 10 times less. The vacuum extraction protocol which is more convenient in dealing with such a large volume of sample was not working well with Zymo Kit (note: I used regular vacuum available in our lab). The recommended centrifugation speed by Zymo kit was also not working well so I increased it and allowed it to go for more time to finish the extraction. Adding these additional steps lead to lots of inconvenience. So I will suggest Qiagen over Zymo. One of my colleague also said that my samples are of better quality then Roche kit so it suggests preparation is good compared to Roche kit as well. I don't have any experience with other cell free DNA kits.
Qiagen circulating nucleic acid kit suggest leaving columns for 2-3 min after adding AVE buffer at the elution step. However, I found that 2-3 min is not enough to take all samples out of the column. My personal experience suggests that if you leave it for about 30-60 min with AVE buffer then extraction is much better. I agree with you that if you just follow what is written in protocol then recovery is too low. Otherwise, 4-5 ml plasma is a good amount to extract enough cfDNA for PCR and NGS. You should get 50-200 ng DNA from 4ml plasma. If you are getting less than you need to work on your protocol.
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