23 replies to this topic

[hercolanium]

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???!!!!

Edited by hercolanium, 15 November 2013 - 01:19 PM.

[jerryshelly1]

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.

[hercolanium]

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

[jerryshelly1]

It is usually standard, but look and see what anti-coagulant is used on the tube (heparin, etc...)

[hercolanium]

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.

[jerryshelly1]

I honestly don't know. EDTA is a good anticoagulant. Maybe try contacting Promega and see if they have any advice.

[hercolanium]

I honestly don't know. EDTA is a good anticoagulant. Maybe try contacting Promega and see if they have any advice.

I have contacted them so many times, they tried to help me with many suggestions but they didn't work out

Thank you for your effort with me, obviously my case is hopless, hail to science X:-(

[jerryshelly1]

It may be worth it to contact another company and see if you can try one of there kits for free. Since you regularly isolate blood, it will be beneficial for them. I have had good success with EZ Bioresearch's MW blood isolation kit (competitively priced).

 

It makes me worried that two people have commented on the inefficiency of this kit.

[hercolanium]

It may be worth it to contact another company and see if you can try one of there kits for free. Since you regularly isolate blood, it will be beneficial for them. I have had good success with EZ Bioresearch's MW blood isolation kit (competitively priced).

 

It makes me worried that two people have commented on the inefficiency of this kit.

It works perfectly for the the day old samples, but anything older than that gives varying results, which includes some horrible results, and sometimes I cannot go to the lab and extract DNA each time I get a sample, so I need 2-3 days to have full patch since the lab is far.

I cannot change the kit now because I have included it in the thesis proposal and IRB forms

Thank you very much for your effort

Edited by hercolanium, 18 November 2013 - 11:29 AM.