20.109(F20):M1D6

20.109(F20): Laboratory Fundamentals of Biological Engineering

Fall 2020 schedule FYI Assignments Homework Communication | Accessibility

M1: Genomic instability M2: Drug discovery M3: Metabolic engineering

Introduction

Remove your CometChip from the 1X PBS and use a kimwipe to dry the GelBond side.
Carefully move your CometChip to the gel electrophoresis station in the 4 °C cold room.
Place your CometChip on the raised center region of an electrophoresis box.
- Double-sided tape was applied to the gel electrophoresis box. Be sure you lay your CometChip on the tape strips and lightly press down with a pipet tip to ensure it is secure.
Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
Leave the CometChip in the alkaline electrophoresis buffer (aka unwinding buffer) for 45 minutes.
To separate the damaged DNA into 'comets', electrophorese for 40 min (at 16 V, or 1 V/cm).
- It is important that the electrophoresis occur at 300 mA. To maintain the appropriate current, the volume of electrophoresis buffer may need to be adjusted. The teaching faculty will assist you in adding/removing electrophoresis buffer such that this value is reached.
Carefully remove your CometChip from the electrophoresis box and place it in a dish.
Obtain an aliquot of neutralization buffer from the front laboratory bench.
Wash your CometChip by adding enough neutralization buffer to cover (~10-12 mL) and incubate for 5 min at room temperature.
- Repeat this step for a total of 3 washes.
Add the SYBR gold DNA stain to your CometChip and carefully move it to the 4 °C cooler.

The teaching faculty will image your CometChip and provide the images to you in the next laboratory section.

alkaline electrophoresis solution: 0.3 M NaOH, 1 mM Na₂EDTA, pH 13.5 (from Sigma)
neutralization buffer: 0.4 M Tris, pH 7.5 (from Sigma)
SYBR gold DNA stain (from ThermoFisher Scientific)