I am performing a gDNA extraction for sgRNA sequencing and to maintain representation I am required to use large sample size (around 30e6 cells/sample). I have tried some kits that suppose to handle this sample size but all failed at extracting sufficient and good quality DNA. I have then decided to perform Phenol:Chloroform:Isoamyl (PCI) alcohol extraction but with no success since the solution (after lysis and mixing with PCI) was too viscous and though two phases were obtained, the liquid phase had much of the viscous (clear) content.
Did anyone had such issue while performing PCI extraction? can anyone advice how to determine volumes of sample to PCI?
I look forward to any answers,
Thank you.
Dear Dimitri,
Before to answer, could you precise what kind of samples do you have ? plant ? animal culture cells ?
Hello Dmitri,
For eDNA extraction we have a protocol that requires PCI at a ratio of 25:24:1 v/v. It is commercially available in most big chemical suppliers. In our protocols we use 1 volume of the mixture and after mixing we centrifuge to get complete phase separation.
Hope this helps!
Hello Jessica and Gael,
Thank you for the reply, I am using mammalian cell line (DU145)
I appreciate your answers, I have figured my issue with my precipitation.
When the sample was mixed with the PCI and white turbid solution was observed I had to spin the mixture at higher speed and for longer time to achieve the complete phase separation.
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