Regular PCR
Reagent:
- Taq DNA Polymerase from any favorite company.
- dNTP from Invotrogen #10297-018 (250 ul each, 100 mM)
- I usually mix the dNTP by myself using the four separate dNTPS from invitrogen. I use RNase-free H2O to make 10 fold dilutions and freeze aliquots of 50 ul into -20C. This dNTP which is 10 mM each can be used for reverse transcription, molecular cloning and also regular PCR.
- The primers used are usually 10 uM.
- PCR reaction consists of 7 components usually.
1. For regular PCR such as genotying, I try to keep all PCR reactions the same.
H2O: 18.5 ul
dNTP: 0.5 ul
10 X buffer: 2.5 ul
Taq: 0.5 ul
Forward primer: 0.5 ul
Reveres primer: 0.5 ul
Template: 2 ul
-------------------
Total : 25 ul
2. Since the PCR primers are designed using the same criteria, the PCR reaction can be performed similarly.
94C: 5 min
94C: 30 sec
58C: 30 sec
72C: 30 sec
Repeat < 35 cycles
72C: 5 min
4C: forever
The duration of 72C entension can be calculated based on 1000 bp/min.
New feature:
You can add comments on protocols now. Just go to any individual method page and click on
the "Add a comment" link.
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.
...
Read more highlights.
