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.
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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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