Uv spectrophotometer

Origami2 (DE3) E. coli cells transformed with pETHSA and cotransformed with pETHSA-pGro7, pETHSA-pTf16 were grown at 37 °C with and without induction at 220 rpm in shake flasks. At various time intervals, 1 mL of aliquots were withdrawn for turbidity measurements at 600 nm using Beckman UV-Spectrophotometer (USA).
0.3–0.6 mg/mL of Arabinose was added for the expression of a chaperone when the O.D reached 0.2–0.5; followed by induction of rHSA at 0.6–1.0 O.D with 100 µM of IPTG (in the case of IPTG based induction). AI is a self-regulated induction strategy where the inducer is already present in the growth medium [19 (link)]. To maintain non-inducing growth conditions lactose was not added in the AI medium. Expression of proteins was checked by running various withdrawn samples on 12% SDS-PAGE. E. coli Origami2 (DE3) strain was used as a negative control in all these studies.
For calculating the specific growth rate constant, µ, the exponential (or logarithmic) growth phase was used during which the rate of increase of cells is proportional to the number of bacteria present at that time. The specific growth rate constant µ was determined using the following equation: $$ln{N}_t-ln{N}_0=\mathit{\mu }(t-t_0)$$ where N_t = number of cells at time ‘t’; N_o = number of cells at time ‘t = 0’; µ = specific growth rate constant; t = time in hours.

Extraction of DNA was done using a Qia-amplification DNA extraction kit supplied by (Qiagen, USA) according to the manufacturer's instructions. The extracted DNA concentration was read at 260 nm on UV-spectrophotometer supplied by (Beckman, USA). Amplification of the mitochondrial DNA by PCR with the appropriate primers for:
The isolated DNA from each sample was used for amplification. The reaction mixture was prepared by adding 12.5 μl of DreamTaqPCR Master Mix, 0.5 μl of forward and reverse primer (10 μM), and 10.5 μl RNase-freeH₂O. Also, 2 μl of DNA template was added. Using a Thermal Cycler (such as LifePro Thermal Cycler, Bioer), PCR was performed with an initial denaturation at 94 °C for 15 min (1 cycle), followed by denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension to 72 °C for 1 min and 30s (30 cycles). The final extension was performed at 72 °C for 7 min and the concentration of mtDNA was determined in ng/μl [42 (link)].

The tissue total proteins were estimated as described by Lowry et al. [46 (link)]. Briefly, the aliquot and Lowry reagent mixture mixed well, and the change in absorbance of purple color chromogen was noted spectrophotometrically (DU 640B, UV-Spectrophotometer, Beckman Coulter Inc., Brea, CA, USA) at a 750 nm wavelength. The results were expressed as mg of protein per ml of the aliquot solution.

Genomic DNA was extracted from PBMCs by commercially available genomic DNA kit (QIAGEN, Germany or INVITROGEN, USA) as per the manufacturer’s instruction. Concentration and purity of genomic DNA were measured by UV spectrophotometer (BeckMan Coulter, USA). The extracted DNA was appropriately coded and stored for further use.