Tail DNA extraction for regular genotyping - an alternative inexpensive method
Reagents
- Proteinase K Solution from Invitrogen #25530-049 (20 mg/ml).
1. Cut tails from animals of 0.5 cm.
2. Prepare 1 X tail lysis buffer, 50 mM KCl, 10 mM Tris.HCl (pH8.3), 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP-40 (v/v), 0.45% Tween-20 (v/v). After sterilization, make 5 ml aliquots and store at -20C or 4C.
3. Prepare working solution by adding proteinase K to 1 X tail lysis buffer. For each tail, I use 197.5 ul lysis buffer with 2.5 ul Proteinase K solution.
4. Incubate at 55 C overnight. I usually put all the tubes into the hybridization glass bottle and keep rotating in the Isotemp incubator.
5. The next morning, vortex the tubes to mix the solution and boil it at 95C for 15 min to inactivate Proteinase K.
6. Spin down at 14K RPM for 3 min.
7. Take 2 ul for genotyping.
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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....
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