Tail DNA extraction for regular genotyping

Reagents

1. Cut tails from animals of 0.5 cm.

2. Prepare tail lysis buffer by adding proteinase K to the Viagen Lysis Reagent. For each tail, I use 250 ul lysis buffer which contains 2.5 ul Proteinase K solution in 247.5 ul Viagen Lysis Reagent.

3. Incubate at 55 C overnight. I usually put all the tubes into the hybridization glass bottle and keep rotating in the Isotemp incubator.

4. The next morning, vortex the tubes to mix the solution and boil it at 85 C for 45 min to inactivate Proteinase K.

5. Spin down at 14k RPM for 3 min.

6. Take 2 ul for genotyping.

7. This DNA preparation is very stable at RT. I have tested it after 1 week which does not affect the quality for genotyping.


Comments

Add a comment
Posted by ebiomethods Mon, 22 Dec 2008 22:02:38
Subject: Comments are welcome
Please give us your feedback to help us improve.
Posted by neverthink (nevernetbug) Tue, 23 Dec 2008 15:40:56
Subject: Another simpler way to get DNA
it would only take Alkaline lysis buffer 15-30 min at 95 degree and then neuralize with Acedic buffer and then go on with PCR, without doing any other steps. one could finish genotyping within 2-3hrs from cutting tail with minimum handling. This is now a very widely used protocol, also commercial kits available but not worth buying though.

Posted by guest Tue, 10 Feb 2009 08:09:57
Subject: please tell us the detailed components of Alkaline lysis and

Posted by ebiomethods Tue, 10 Mar 2009 16:09:34
Subject: reply to guest
Please refer to \\\'Tail DNA extraction for regular genotyping - an alternative inexpensive method\\\' for details.
Site News
New feature:
You can add comments on protocols now. Just go to any individual method page and click on the "Add a comment" link.

Highlight of the month
Autophagy hits heart

Autophagy is acutely upregulated to provide necessary nutrients during starvation for survival. Under normal condition, constitutive autophagy is critical to perform house-keeping functions to eliminate damaged organelles and dysfunctional long-lived proteins.

Ageing is associated with accumulation of dysfunctional proteins in cells. Autophagic activity is decreasing along with ageing. Studies in worms and flies show that reconstitution of autophagy can increase life span. A recent report by Taneike and colleagues explored the role of autophagy in age-related cardiomyopathy.

In heart, autophagic activity is decreasing when ageing. Taneike et al eliminated autophagy in heart by knocking out an essential gene, ATG5, specifically in heart. The researchers found deteriorated cardiac function at 10 months of age in the deficient animals compared to wild type controls. The dysfunction of autophagy causes accumulation of dysfunctional mitochondrial, reduced mitochondrial efficiency and significant oxidative damage in cardiomyocytes. More interestingly, evidence of mitochondrial damage is obvious as early as 3 months old, before cardiac remodeling and dysfunction manifest. This study points to a possibility that autophagy is critical in maintaining cardiomyocyte homeostasis and autophagy may be a target for future therapeutic design for heart failure.... Read more highlights.