Lab 1: RNA Extraction and Protein Analysis, Part 1
Summary:The purpose of this lab was to extract RNA and protein from a tissue sample from a juvenile sockeye salmon using Tri-Reagant and CellLytic MT. The concentration of the protein was then to be determined using the Bradford protein assay. Basic instruction on primer design for selected gene sequences was provided as well.
Materials and Methods:
RNA Extraction
I chose the juvenile sockeye salmon sample that had been subjected to pH treatment C. I first added 500 uL of TriReagent to the 1.5 mL snap cap tube containing my tissue. I homogenized the tissue using a disposable pestle. The tissue was not homogenizing well so I placed on the vortex for about 10 seconds, attempted to homogenize using the pestle again, and then placed on the vortex for approximately 15 seconds. The tissue was still not completely homogenized, but was not breaking down any further. I then added 500 uL of TriReagent and placed on the vortex for 15 seconds then stored at -80°C.
Protein Extraction and Analysis
The sockeye salmon sample that I used for my protein extraction weighed 32mg. I added 500uL of CellLytic MT to the 1.5 mL snap cap tube and homogenized with a disposable pestle. I then inverted the tube several times to ensure mixing. I then placed the tube in a centrifuge and spun for 10 minutes at the highest speed. I then transferred the supernatant liquid into a fresh tube labeled “Protein, sockeye pH C, EJ, 10/05/10” and placed on ice. I then labeled a 2 mL tube with “Protein BA, EJ 10/05/10” and pipetted 15uL of my protein sample and 15uL of DI water into the tube. I created a control tube by pipetting 30uL of DI water in a 2mL tube. I then pipetted 1500uL of Bradford Reagent to each tube, mixed by inversion, and let incubate at room temperature for approximately 10 minutes.I mixed my blank solution via pipetting and transferred 1000uL to a disposable cuvette to zero the spectrophotometer. I then mixed my sample via pipetting and transferred 1000uL into a new cuvette, measured and recorded the absorbance, then repeated to obtain a second absorbance. The protein sample was then stored at -20°C. The concentration was then calculated using the formula y=1013.9x.
Results:
The results of the RNA extraction will be completed on a future date. I had a difficult time homogenizing the sample, so I am hoping there will be enough RNA to perform the future RNA quantification.
The first absorbance result I obtained was .244 and the second was .260, yielding and average of .252. Using the formula provided, y=1013.9x for determining concentration I got the result of .511 (ug/mL).
Y=1013.9x
=1013.9(.252)=255.50
255.50 was then multiplied by 2 to account for the dilution with DI water.
Conclusions? Reflection? Genes of interest? -