Hi,
Need help finding a good protocol for genotyping mice from tail snips. I really need suggestions on how to extract the DNA and have a good sample solution. My DNA in the sample solutions are coming out to be quite low and I can't figure out what I am doing wrong.
Hi,
I have been using this protocol:
Tail DNA isolation Protocol
1. Tail Lysis Solution(TLS)
100mM Tris-HCl pH8.5
5mM EDTA pH8.0
0.2% SDS
200mM NaCl
400ug/ml Proteinase K (Sigma) (20mg/ml 1:20 dilute in TLS)
(Proteinase K must be added freshly to the TLS Buffer)
2. TE solution
10mM Tris-HCl pH8.0
1mM EDTA pH 8.0 (0.1 mM EDTA (); we take 1 mM
* …if you do not see the white DNA clew, incubate 30 min on ice and spin 7 min at 10000 rpm, decant the supernatant and resuspend pellet (sometimes not visible) in 150µl TE-buffer, go on with step 9
Ref: Laird, P.W., et al. NAR, Vol.19, No.15, 4293
This is the quick way of doing it. We used a similar protocol in my previous lab and never had issues with the resulting DNA.
https://www.midwestern.edu/Documents/Faculty%20Web%20Pages/NaOH%20Tail%20Prep%20and%20PCR.pdf
I attached the protocol we use.
The digest buffer is propitiatory so I dont know exactly whats in it.
But I do dilute it one to one with molecular grade water to make it last twice as long. We use proteinase-K with this digest buffer and always get good dna for PCR.
Best Wishes!
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