DNA degradation - (Sep/20/2006 )
Hi,
I extract bloods using phenol chloroform, but recently i have noticed a serious degradation problem with the resulting DNA- when i run it on a gel it looks like a ladder!! the laddering is quite regular, which made me suspect exonuclease activity;
so i made up all the buffers a fresh and was extra careful, however when i ran it out on a gel the same problem occured. The same problem is occuring with some of my collgues but not all of them.
Any ideas how this might be happening?
I have attached the protocol i used.
Thanks
I extract bloods using phenol chloroform, but recently i have noticed a serious degradation problem with the resulting DNA- when i run it on a gel it looks like a ladder!! the laddering is quite regular, which made me suspect exonuclease activity;
Perhaps a problem in lysis, you leave cells at room temperature, then lyse them without chemical that kills nucleases (SDS, Proteinase) or their activity (EDTA). So perhaps enough time for the nucleases? Phenol comes first in step 6.8. so perhaps too late.
Like a person with only a hammer for a tool, I would suggest giving a guanidine thiocyanate base lysis solution a go.
The guandine and thiocyanate ions denature proteins and lyse the cells, the EDTA in it chelate any divalent ions. And the detergent in the mixture also helps cell lysis and helps a little to denature the proteins. THe cell's polysacharide clump together and can be spun away while the DNA remains in solution until percipitated by EtOH.
The DNA that one gets at the end needs alittle RNAse and phenol. But nothing major.
Hmmm... this seems to be my answer to any genomic extraction problem. I guess I like this solution very much.
As for the nuclease problem... well... keeping your work on constantly on ice help. A cold centrifuge is a must. And I hear that using refrigerator cooled buffers might help.
Thanks for your help. i will give it a try.
The guandine and thiocyanate ions denature proteins and lyse the cells, the EDTA in it chelate any divalent ions. And the detergent in the mixture also helps cell lysis and helps a little to denature the proteins. THe cell's polysacharide clump together and can be spun away while the DNA remains in solution until percipitated by EtOH.
The DNA that one gets at the end needs alittle RNAse and phenol. But nothing major.
Could you direct me to a protocol, i am having a bit of trouble pinning one down, thanks
4M Guanidine Thiocynate 47.264g
17% Isopropanol 17ml
0.1M Sodium Acetate 1.3608g
0.1M 2-Aminethanethiol. Hydrochloride 1.1361g
0.2% Sarkosyl (Sodium lauroyl Sarcosinate) 0.2g
10mM EDTA 0.37224g
30mM Citrate 0.8823g
adjust to pH 8.5
Total volume 100ml
This is just one variation on a theme. I have seen variation in the amount of guadinie thiocyanate used (3.8M to 4M), the amount of sarkosyl (0.2 to 0.5%) and the amount of EDTA. Some variations add phenol and use glycerol to solubalise the two.
This is my variation. Thinker with it and maybe it'll work better.
Thanks thats great. I really appreciate it.
Just to check, this solution will lyse the nuclei so i want to keep the supernant and disgard the pellet (i just want to check as this is opposite to my protocol, where we lyse the cells but spin down to keep the nuclei, and at a later step lyse the nuclei).
Thanks for alll your help