DNA extraction impurities - (Nov/02/2013 )
jerryshelly1 on Tue Nov 5 18:31:24 2013 said:
It may affect your DNA concentration, but not the residual protein. If they are immune compromised (i.e. low WBC's) you would expect a lower than average DNA concentration, but the overall consistency of the blood would be similar.
As far as the chemotherapy, I do not have enough knowledge on that to make a guess. Are you only seeing this phenomenon in you cancer and chemo patients? Do your healthy controls show this?
I checked the wizard protocol and it is very straight forward. Do you see any clumps in the whole blood before you begin your extraction? Is this a recent problem that was absent in the past?
Thanks for your thorough reply.
I've faced this problem since the beginning with both cases and controls, there were no clumps in the blood and the same problem happened when I extracted the DNA from
samples drawn on the same day by professionals.
The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.
Maybe...
Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?
jerryshelly1 on Tue Nov 5 19:44:01 2013 said:
The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.
Maybe...
Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?
I've tried all that except precipitation at 4C for 10, I'll try it tomorrow
thanks
Hi, I am having the same problem so I have tried your instructions by incubating the samples at 4 C with the Nuclei lysis solution and Protein Precipitation solution, the 1-2 days old samples came out bloody and impure, the DNA pellet came out brown like it has been burned after transfering to isopropanol tube and centrifugation
Please help 
PM Sayk and see if he/she worked out the problem.
jerryshelly1 on Thu Nov 14 15:20:50 2013 said:
PM Sayk and see if he/she worked out the problem.
Uhm, I have asked her about it and it only worked out for her on the one day old samples, the older ones; say 2-3 days still came out bloody...The DNA pellet which should be white came out a dark brown like it was burned. I use 950 ul Cell Lysis Solution, 350 ul for all of Nuceli Lysis Solution, Isopropanol and 70% Alcohol, and 150 ul Protein Precipitation Solution. I have increased the centrifugation times and I have done the incubation step you pointed (Nuceli Lysis Solution+Protein Precipitation Solution at 4 C)
What should I do???!!!! 
What kit are you using? Make sure all your solutions are homogenous. If you continue to have problems, you can contact the manufacturer and see if there have been additional problems with your particular lot number. If anything, they may give you a new kit. I'm not certain though.
jerryshelly1 on Sun Nov 17 01:15:48 2013 said:
What kit are you using? Make sure all your solutions are homogenous. If you continue to have problems, you can contact the manufacturer and see if there have been additional problems with your particular lot number. If anything, they may give you a new kit. I'm not certain though.
I am using Promega Wizard DNA Extraction Kit. The solutions are new and homogenous, the problem is that new samples turn out good while old sample vary between even dupliicates of the same sample
I will try again this week to see what happens and gat back at you
Thanks and take care
It is usually standard, but look and see what anti-coagulant is used on the tube (heparin, etc...)
jerryshelly1 on Sun Nov 17 17:57:55 2013 said:
It is usually standard, but look and see what anti-coagulant is used on the tube (heparin, etc...)
I am using a purple top EDTA tube, I take 2.5 ml and do duplicated of each sample.