May 8, 2005 at 3:28 pm #865saugagirlParticipant
Hi everyone! If anyone out there is familiar with the kiwi DNA extraction protocols, maybe you could help answer a few qestions please?
First of all, why do most protocols recommend a temp. of 60 degrees? I understand that the temp. must be high to denature the enzymes and proteins that could break down the DNA. However, we were told NOT to exceed this temp. I don’t really understand why; shouldn’t a hotter temp. denature more enzymes, and provide better observations of the DNA?
Also, our group used ethanol, but isopropyl was another option. After the lab, the instructor told us to speculate the effects of using the other alcohol instead of whatever each group used. Should there be a difference? I thought the purpose of the alcohol was to causes the DNA to precipitate, so wouldn’t both do this? Or would one result in more DNA than the other? I read somewhere on the net that isopropyl alcohol is oxidized more slowly than ethanol…. but I would appreciate some help in this area as I’m not really familiar with the effects of their chemical properties.
Finally, when we performed the lab, we tried out three different methods of adding the kiwi/salt/water/detergent solution to the ethanol. I hypothesized that there shouldn’t be a major different in the amount of DNA precipitate… but suprise!! That’s not what we saw.
A- kiwi solution added first, then ethanol gently added on top (no stirring). This gave the least DNA.
B- kiwi first, then ethanol STIRRED in. This gave a fair amount of DNA.
C- ethanol first, then kiwi gently on top, no stirring. This had the most DNA.
Any ideas as to why this happened? I imagine it has to do with DNA’s lack of solubility in ethanol (and I believe it’s less dense as well), but…. each test tube had ethanol in it in the end! Why was there a difference at all?
Thank you for reading this far. 😀 I’d really appreciate ANY help!
June 2, 2005 at 7:26 pm #23604b_d_41501Participant
Exceeding this temperature would not only denature the enzymes, but the DNA you are trying to extract as well. Thus, you do not want this to occur. The alcohol is used to help precipitate the DNA, however, different alcohols may have different effects. Ethanol was always used in my labs. I’m assuming (AKA my opinion) that since the ethanol was on the bottom the kiwi solution gently filtered through the ethanol allowing the DNA to filter as you hypothesized in your post.
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