Plasmid miniprep - Eppendorf
Reagents
- Miniprep Kit from Eppendorf #2300000 (X100).
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 5000 RPM for 5 min at RT.
3. Remove supernatant and resuspend the pellet thoroughly with 400 ul lysis buffer (keep at 4C with RNase added).
4. Incubate for 1 min at RT.
5. Spin down at 14K RPM for 1 min at RT. Keep the sample in the centrifuger and add 400 ul washing buffer (isopropanol added).
6. Spin down at 14K RPM for 1 min at RT. Discard flowthrough.
7. Spin down one more time at 14K RPM for 1 min at RT.
8. 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.
The DNA prepared can be used for sequencing or enzymatic digestion.
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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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