I am using a standard phenol chloroform extraction protocol to extract DNA from very small amounts of insect tissue. I have included extra steps (KAc treatment prior to phenol step) to remove additional proteins. However, during the subsequent ethanol precipitation two pellets are observed for each sample rather than the usual one. One of the pellets is dark in colour (brownish) and the other is white. Firstly, has anyone ever seen this before? I had not and assumed that the DNA would be in the white pellet and the dark pellet would be excess proteins. Small amount of experimentation shows that it is the dark pellet that contains the DNA. Spectrophotometry readings are in the range of 1.7-1.8 for the 260/280 and around 1.2 for the 260/230. Not absolutely awful but not brilliant either. This seems to indicate there is still protein in this dark pellet. Does anyone have any suggestions as to what I can do to further "clean" this dark pellet or to modify my protocol?
Hey thanks for responding, I did 2 x 70% ethanol washes and both pellets are still visible after each spin.
I have also tried adding an ammonium acetate precipitation step (supposed to remove sugars and such) prior to the phenol stage and this didn't help either. In fact I lost quite a bit of the DNA doing that too.
Hi Nicola, Have you considered using a kit? I used to do my insect DNA extractions by the phenol/chloroform method but now routinely use commercial kits which are great value and yield high quality DNA. I can recommend the Qiagen DNesyKit (69504) or the Zymo Research ZR Tissue and Insect DNA Kit (D6016). Both of these kits work well for small insect specimens e.g. weevils. I usually use two legs or the head capsule (ending up with a 100ul volume of approx. 100ng/ul DNA). My favourite is the Qiagen kit because the yields are better but if money is tight then the Zymo kit is much cheaper (and you can increase its yield by adding proteinase K digestion step). Good luck Nicky
Hi Nicola, are you using 1:1 phenol/chloroform? If not, try this. And: how is your phenol? It shouldn't be more than one year old (storage in a fridge?). Is it still transparent (no color)? Cheers, Nadine
Hi Nicola, thanks for your input - I was originally using QIAgen kits before (following manufacturer's instructions for extraction from insect tissue) and found that the DNA was of low yield and very poor quality - 260/280 ratios of around 1.2!! Lets not even discuss the 260/230 lol! I think this may be due to the small amounts of tissue I am extracting from - head capsules of very tiny parasitoid wasps. Could you recommend any further additions to the protocol I might try, I still have kit available (I already carry out a 3-4 hour ProtK digestion when using the kit)? I find that the phenol:chloroform is still better that the kits even with the problem I am having - 260/280 = c. 1.6-1.7. The DNA PCR's ok but I would prefer it to be cleaner for NGS etc.
Hi Nadine, I have been using phenol:chloroform:isoamyl alcohol 25:24:1 and using this mixture in a 1:1 ratio with the sample. We store the phenol in the fridge and the 24:1 chloroform:isoamyl at room temp. The phenol has 8-hydroxyquinoline added so the phenol appears yellow all the time.
Thanks again for your input this is a puzzle :-/
Hi Nicola, i am planning to use phenol choloroform method to precipitate my rna. i did purchase pre made phenol: cholorophorm: isoamyl solution, and one step require me to put chlorophorm isoamyl only. so my question is how do you separate and make sure that you are taking chlorophorm:isoamyl part from the solution? your previous post showed that u use chlorophorm isoamyl. thanks
Hi Nor,
We stock all the chemicals separately so that I can make up the solutions that we require. you wont be able to separate out the phenol the chlororform and the iso-amyl from the stuff that you have. sorry!
Nicki
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