questions about isolating genomic DNA by alkaline - (Nov/22/2008 )
Hi all,
I am a pharmacology student trying to isolating genomic DNA by alkaline.
But I just abstract protein...
I worked with tail snips from adult mice 6weeks old,are these mice too old for NaOH alkaline hydrolysis?
and I don't know the principle well.We know that DNA will be dameged at high temperature,but,I found a DNA abstract method named HotSHOT(hydrolysis at 90 degree centigrade for more than 30min),genomic DNA abstracted in this method can be used for PCR.Isn't it denatured?
-meredith
The DNA extracted by alkaline lysis is not a pure extraction by any means, but it is definitely good enough for genotyping. During the process make sure that you follow the conditions carefully, including being very careful not to transfer any of the precipitated protein into the fresh tube after the protein precipitation and centrifuge step.
If you are using Ammonium or Sodium Acetate, be aware that these decay with time in solution so you need to have relatively fresh solutions (less than 3 months is best) and that they are not interchangeable directly, you will need more ammonium acetate to precipitate the protein and DNA than you will of the sodium salt.
Hello bob1,
Thank you for your suggestion.
I repeat the extraction these two days,and I found that when adding less tail tissue there are less protein remain.
But when I prolong the lysis time to 40min, it seems there is less DNA isolated than 30min(judged by fluorescence intensity after electrophoresis ).Dose degradation happen?
and,what do you mean by"is not a pure extraction"?What are the predominant impurities?RNA?Protein?
-meredith
Pure as in DNA only, there will be lots of protein and RNA left behind in an alkaline lysis extraction such as is used for tail tips.
Yes the DNA can degrade, there are lots of DNases released when you rupture the cells.
Yes the DNA can degrade, there are lots of DNases released when you rupture the cells.
I run PCR with DNA isolated in this way,and seems to get the expected band.
But there are several non-specificity bands,and lots of RNA dimer,I will try to optimize the experiment.
Thank you.
[attachment=5609:081126pcr_2_.JPG]
Nice to see it is working. I wouldn't have said that there are any RNA dimers in there, it will be DNA smear, not RNA. You do have some primer dimers though.
Sorry,they are primer dimers.