BCH 369L: Biochemistry Lab Report

BCH 369L–Biochemistry Laboratory Fall 2016
Module C
Orlando Martinez uteid: olm298

Introduction
The purpose of this lab is to implement the technique of gel electrophoresis in the purification and size determination of various proteins and DNA fragments. In order to do this, a polyacrylamide gel will be prepared and placed in a buffer-containing gel electrophoresis apparatus. Next, an aliquot of acid phosphatase and a molecular weight marker (Composed of Phosphorylase B, bovine serum albumin, ovalbumin, and carbonic anhydrase) will be placed into separate wells within the gel, and the apparatus will be connected to a constant voltage source (175 V) for an allotted period of time (60 min). This will allow electricity to flow through the apparatus,
Spacers Form cavity within gel mold
Plastic bag Holds gel/ glass combination during liquid gel pour
Comb Forms wells within gels
Gel casting stand Holds gel mold in place during gel transfer
Ammonium Persulfate Reacts with TEMED to form polyacrylamide gel
Tetramethyethylenediamene (TEMED) Reacts with ammonium persulfate to form polyacrylamide gel
Vertical electrophoresis apparatus Contains running buffer and polyacrylamide gel during electrophoresis
1X Page running buffer Maintains pH in electrophoresis apparatus and transfers current
Voltage and current regulator Electricity source
Acid phosphatase Protein analyzed during gel electrophoresis
Molecular weight marker Used to calculate protein molecular weight
Extra-long gel loading pipette tips Used to transfer proteins/DNA to gels
Coomasie blue stain Used to stain protein bands
Horizontal gel apparatus Contains buffer and agarose gel during gel electrophoresis
0.7% Agarose in 1X TBE and 0.005 ethidium bromide Gel used in DNA electrophoresis
1X TBE Buffer Maintains pH in electrophoresis apparatus and transfers current
HindIII digested lambda DNA DNA analyzed by gel electrophoresis
EcoR1 lambda DNA DNA analyzed by gel electrophoresis
1 kb DNA ladder Used to estimate DNA fragments’
This was done by melting a solution composed of 0.7% agarose in 1x TBE and 0.005% ethidium bromide, and pouring the resulting liquid into comb-containing gel tray until the thickness reached about 6 mm. Once the gel had been allowed to cool for about 20 minutes, it was removed from the tray, placed in a horizontal gel apparatus, and immersed in 1x TBE buffer. Next, three different solutions were prepared from an aliquot of HindII digested lambda DNA and 3 microliters of 6x loading dye, an aliquot of EcoRI digested lambda DNA and 3 microliters of 6x loading dye, and 1 kb DNA ladder and 3 microliters of 6x loading dye. The resulting solutions were then transferred into separate wells within the gel, and the apparatus was connected to a constant current source (45mA) for 60 minutes. After producing clearly visible bands, the gel was taken out of the apparatus and photographed with an ultraviolet light box (Displayed in Figure