Plasmid miniprep - Qiagen
Reagents
- Miniprep kit from Qiagen #27106.
- Keep P1 solution at 4C after RNease added.
The DNA prepared can be used for sequencing, enzymatic digestion, subcloning, etc.
1. Day 1, seed bacteria to 3 ml LB liquid medium and culture overnight at 37C, RPM 220.
2. Day 2, transfer 1.5 ml bacteria to tubes and spin down at 5K RPM for 5 min at RT.
3. Remove supernatant and resuspend the pellet thoroughly with 250 ul P1 solution.
4. Add 250 ul buffer P2 and invert the tube 2-3 times gently. The solution becomes clear.
5. Add 350 ul buffer N3 and invert the tube 2-3 times gently. Spin down at 14000 RPM for 10 min at RT.
6. Carefully transfer the supernatant to a column. Spin down at 14K RPM for 1 min at RT. Discard flowthrough.
7. Apply 500 ul buffer PB and spin down at 14K RPM for 1 min at RT. Discard flowthrough.
8. Apply 700 ul buffer PE (with ethanol added). Spin down at 14K RPM for 1 min at RT. Discard flowthrough.
9. Spin down one more time at 14K RPM for 1 min at RT.
10. Transfer the column to a clean tube. Apply 30-50 ul TE buffer to the center of the column.Incubate for 1 min at RT.
11. Spin down to collect DNA at 14K RPM for 1 min at RT.
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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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