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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SIRT6 links glucose and lipid metabolism
SIRT1, a histone deacetylase, has drawn considerable attention due to its important roles in metabolic regulation and longevity. SIRT1 is induced by fasting and suppressed by feeding. SIRT1 increases glucose production and beta oxidation in liver to meet energy needs during food deprivation. Another member of SIRT family, SIRT6 follows similar expression patterns as SIRT1. The function of SIRT6 in metabolic regulation remains unknown.
Kim and colleagues found SIRT1 can regulate SIRT6 expression in liver. Specifically, SIRT1 functions together with FOXO3a and stimulates SIRT6 transcription during fasting. The activation of SIRT6 directly suppresses gene expression of metabolic enzymes involved in triglyceride synthesis and glycolysis in liver. Deficiency of SIRT6 causes increases of glucose utilization, reduced beta oxidation and as a result, fatty liver. More importantly, Kim et al found the expression of SIRT6 is decreased in human fatty liver samples which indicates SIRT6 may play critical roles in liver steatosis in clinical settings. This study landed in Cell Metabolism of this month.
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