What is the best method for preparation freshly Saturated phenol or Unsaturated phenol, and what's different?

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.

I searching for information about the use of phenol solution in nucleic acids extraction. Phenol with low pH (let say pH = 4.5) seems to be better fitted to extract RNA. Is there any difference if acid-phenol contains a buffer, i.e. citrate buffer? To what extend does the pH influence the extraction of RNA and DNA? What about water-saturated phenol? Often, there is a buffered solution provided with phenol solutions. When is it useful to add it to the solution? Does the same apply to PCI?

I really appreciate your help.

Goodevening!

I am currently optimizing stimulation time for RANKL induced RAW-Osteoclast for qPCR analysis.

for osteclast markers such as TRAP, MMP-9, Cathepsin K, a significant mRNA fold increase (about 40fold increase and above) was observed with RANKL stimulation of 2 days.

however, for osteoclast transcription factors such as NFATc1, and cfos, the fold increase was very minimal (about 1.02) or even less than the control group.

stimulating RAW cells with RANKL 50ng/mL for 3 days or 4 days did increased cfos transcription level by 1.2.

however, I can't see any increase of NFATc1.

Has anyone of you had experienced similar problem?

here is my protocol.

seeding RAW 264.7 cell in 6 well. 2 x10^4 cell/ml 2mL/well

culture for 24 hour.

then refresh media. stimulate with RANKL (50ng/mL final concentration)

for 2,3, or 4 days.

media was refreshed every 2 days

on day 4 , I have seen few multinucleated osteoclasts.

should i change the media in to absolutly phenol red free media? or re-design my primer? the melt curve was very clean though...

Hi everyone, I did gene cloning using topo TA vector up to the mini prep step, then I cleaned the samples using phenol: chloroform, even with the cleaning I got high concentration and a high A260/A280 about 2.035. I digested it with ECORI and ran gel and I didn’t observe bands. What do you recommend to get my sample to be more of pure? Do I perform spin colum purification? Thanks,

i want to react silanol with phenol.can any one tell me that this reaction is possible and under what conditions?

Hi everyone,

I am trying to generate Dendritic cells from mouse bone marrow where I am culturing the bone marrow cells with GM-CSF for 6 days and measuring the percent DCs generated on 6th day by looking for CD11c positive cells by flow cytometry. But the yield I am getting of DCs is only 60-70%.Is there any other method of DC development from mouse bone arrow which can give better yield? Should I use GM-CSF+IL-4 instead of only GM-CSF for culturing mouse bone marrow cells?

Our group is trying to measure ATP in tissue. Phenol is used in the protocol. We were able to get a good reading, but are having trouble measuring protein content in the same lysate. We believe the phenol may react with BCA reagent. We tried the Bradford method and it still didn't work. Any suggestion?

I'd like to pimp my thesis/oral presentations/next publication with some current facts on implant failure/insufficient osseointegration rates to emphasize the need for new adaptive materials in the field. Anyone got some numbers on that?

I want to model and analysis bone in Abaqus. I studied papers and they use "Mimics" software for it; however, I can't access to source of Mimics. I don't find source of this software in the internet.  Does anybody has the source of Mimics? or Does anybody has any suggestion about modeling bone with another software? My friends work with 3D-DOCTOR Software too and they have problem with capacity of output file for Abaqus or Ansys. the capacity of output file is big. 

Thanks