After several protocols for gDNA isolation i have noticed that in all samples teh DNA is totally degraded.
I have used several papers, some of them are:
https://www.researchgate.net/publication/304540047_Efficiency_of_Different_DNA_Extraction_Methods_for_Fish_Tissues_A_Comparative_Analysis , focusing on the SNET method, or according this paper:
https://academic.oup.com/nar/article/25/22/4692/2361305/Universal-and-rapid-salt-extraction-of-high, etc...
Please see some pictures attached, in some of them I have some plant DNA as compassion, isolated with same chemicals and stock solutions required for the working lysis buffers.
So basically, CTAB protocols, based on phenol -chloroform isoamyl alchohol were mostly used . Most of the time the sample were incubated overnight at 55C with lysis buffer (100-200mg with 900ul buffer), and most of the protocols required this overnight incubation , later on precipitated either with izopropanol or different concentration of ethanol (70-100%).
Some fish samples are store for 6 months, so to be sure to have intact sample, we have used also a fresh fish from market, but no success. Same degradation pattern.
Any suggestions?
Article Efficiency of Different DNA Extraction Methods for Fish Tiss...
First picture, the first four samples are fish , and the remaining three samples are onions. In the second picture, LUK is onion sample, and R1 is fish.
hello Jasmin,
you do not say if the plant dna was processed at exactly the same time as the fish dna. If not then they are not a true control of the reagents and method.
Your picture does not look like degraded dna to me.It looks like no DNA and much RNA. That strong lower band may be RNA .Degraded DNA looks more like a smear on agarose gels. Measure the OD260/280 and 230 ratios to determine if your product is dna or rna.
Phenol is acidic naturally and the use of phenol to purify either DNA or Rna depends very strongly on the pH of your phenol. If your phenol has become acidic then the RNA will stay in the aqueous layer and the DNA will stick in the interface between the 2 layers after centrifugation. I attach a paper which is very informative. Try either new phenol (best) or thoroughly shaking your phenol with stronger tris ph8 to get rid of any residual acidity because the molarity of your TE is quite low and may be overwhelmed by the phenol. Then equilibrate again with your TE ( pH tested also) again. Also measure the pH of the TE that the phenol is equilibrating in, It must be alkaline. Then try the dna prep in parallel with onion dna prep again. Good luck,
Paul
Dear Paul,
Thanks for the note. However, trying few protocols, mainly with following buffer (50mM TrisHcl, 250mM NaCl, 100mM EDTA), later including 30ul of 20% SDS and 30ul of 20mg/ml of Proteinase K into each sample (600ul of buffer). After incubation at 55C we added equal volume of Phenol: Chloroform: Izoamy alcohol 25:24: 1, an already made solution , from Sigma Aldrich, twice. Precipitation was done with 100% ethanol, and 3M Sodium acetate. The pellet was visible as soon a put ice cold ethanol. After i washed it with cold 70% ethanol and dissolved it in 100-200ul TE buffer.
Maybe the SDS is a problem? I tried with frozen wish, with fish stored in ethanol, with fresh fish, but always we got bands that looks like RNA. The same Phenol: Chloroform: Izoamy alcohol 25:24: 1 is used for plant seed isolation and we got the DNA.
It seems my DNA gets lost somewhere in the steps ...:(...
hi Jasmin,
I do not think that the sds is a problem. It is very stable and it helps proteinase K by opening up the proteins for the proteinase to degrade but proteinaseK overnight will degrade the protein on its own. both RNA and salts can precipitate in 70% salt ethanol so it is still possible that the DNA is not there to precipitate, Even commercial phenol preparations with quinolone and other stabilisers will age and become acidic so may not work as expected. The information sheets that come with Trizol type preparations are usually very informative with respect to problems which may occur , .I think it is worth borrowing some from another lab/researcher .If using the same tissue disruption method for seeds and fish it is possible that as fish is soft tissue then the sample may be degraded by heat or mechanical action so you could try a softer or shorter mechanical disruption , It would still be useful to have an OD ratio as that might point to where the process is not working.Check the colour of your phenol also. If it is turning yellow rather than clear or pink that is not a good sign
Thanks Paul, I have checked my phenol, it looks that the phenol i have (mixture with chloroform izoamyl alcohol is) is yellow. Should it be with the combination of chloroform izoamyl alcohol this much yellow ? See the picture ..
this is a terrible colour Jasmin. I think it is past saving by equilibratiEg with alkaline TE and the best ting is to buy or borrow some fresh phenol. When you get it you may want to store some aliquots frozen at -20c as it is more stable over long periods frozen than at room temperature. The yellow colour indicates acidity and the phenol will not behave as you expect in DNA or RNA preparation. Pure phenol is colourless and some commercial phenols have dye indicator added to show acidity and these are usually pink so pink or clear is good and straw coloured is bad
Thanks, according the bottle indicator it is stored at plus 4, it was all the time in brown bottle. I will try Paul what you proposed. Thanks again. I will inform you
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