DNA end blunting
Reagents
- T4 DNA polymerase from Promega #M421A (100U).
- dNTP from Invotrogen #10297-018 (250 ul each, 100 mM).
DNA end blunting is a very common approach in molecular cloning. Although the advances in PCR technology make it easy to engineer any restriction enzymatic sites, straight cloning is still the first choice whenever possible. Adding restriction sites by PCR usually requires an extra sequencing step to make sure no mutations are introduced. In straight cloning, some time only one restriction site is compatible. Then one end sticky, one end blunt approach can be tried. Under some extreme conditions, double blunt cloning is required with both ends are blunt.
For one end sticky, one end blunt approach:
1. For example, EcoR I can be used as sticky end and Sal I can be used for blunting.
2. Digest 1 ug DNA with Sal I to complete.
3. Clean the DNA and elute DNA with 30 ul TE buffer.
4. Add 3 ul 10 X polymerase buffer, 1 ul T4 DNA polymerase, 0.6 ul BSA (Promega), 0.6 ul dNTP (10 mM each) and mix.
5. Incubate at 37C for 1 hr.
6. Clean the DNA and elute DNA with 30 ul TE buffer.
7. Digest with EcoR I to complete.
8. Either clean the DNA for ligation or extract the right band for ligation.
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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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