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DNA extraction from mouse tail


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#1 lukceg1983

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Posted 12 March 2010 - 04:39 PM

Hello everybody

I am beginner at mouse genotyping. I have problems to extract DNA from the mouse tail sample. It's strange because protocol is very simple and it is routinely used in my lab. Here it is:
1. Obtain tail biopsies: 0.2 cm
2. Add 500 microliters of tail lysis buffer with sds + Proteinase K (10 mg/ml).
3. Incubate overnight at 55 degrees C.
4. Spin 2 min 14.000xg
5. Take 350 microliter to new tube
6. Add 1 vol isopropanol
7. spin 3 min 14.000xg
8. Wash with 70% etoh
9. spin and remove etoh
10. dry shortly
11. resuspend in TE buffer (150 microliter), incubate 30 min at 55 degree

After this procedure I don't have dna in my sample or very very little. Digestion seems to be ok. I am loosing my DNA somehow during extraction but don't know at which step. After centrifugation with isopropanol pelet is already very small. What do you think? Has anybody experianced similar problem. I would be very grateful for fast help. Please do it for my poor mice.
thank you in advance

#2 bob1

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Posted 15 March 2010 - 04:00 PM

The protocol looks pretty standard apart from usually there is a step to precipitate the protein before the DNA precipitation - add 1/10th volume of 5 M Na acetate and spin down precipitate. You can also take more of the supernatant after the lysis step too, it works better if you have the maximum amount of DNA obtainable. Usually I have found that inexperienced people tend to touch the pellet while removing supernatant, resulting in the pellet being removed at the same time. You can also do longer spin steps, it should pellet more of the DNA; cooling the precipitation step to -20 deg C should help too.

#3 lukceg1983

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Posted 19 April 2010 - 12:49 PM

The protocol looks pretty standard apart from usually there is a step to precipitate the protein before the DNA precipitation - add 1/10th volume of 5 M Na acetate and spin down precipitate. You can also take more of the supernatant after the lysis step too, it works better if you have the maximum amount of DNA obtainable. Usually I have found that inexperienced people tend to touch the pellet while removing supernatant, resulting in the pellet being removed at the same time. You can also do longer spin steps, it should pellet more of the DNA; cooling the precipitation step to -20 deg C should help too.


Thank you Bob very much for the answer. Yes, I was also concerned about protein precipitation step, but in our lab my colleagues routinely do genotyping without this step and it works well for them. I repeated tail DNA isolation (third time - poor mice) again under supervision of my colleague and using her buffers and it worked out this time:). I think problem was with digestion (something wrong with tail buffer or proteinase K). Two first times tails were not digested completly and the last time digestion was complete (only hair left) and I isolated enough DNA to do genotyping luckily:) Thanks again
cheers
luaksz cegielkowski




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