BIOL 301 Lab Muhammad Fazal
1. Preparing a Total Extract
Add 200 ul of Lysis Buffer to the tube containing the yeast pellet and vortex until cells are
completely resuspended in the buffer.
Pour all the beads (200 ul) into the previous suspension
Vortex the tube for 1 minute at high
Place on ice for 1 minute. (Always keep on ice from now on)
Repeat steps 3 and 4, 7 times
Label, place them in the centrifuge
Spin for 5 minutes at 2000 rpm at 4C
Transfer supernatant (200 ul) to a new tube.
Add 6 ul RNase/DNase to the supernatant
Vortex at 5 for 30 seconds
Incubate on ice for 10 minutes
Add 300 ul of Extraction Buffer and invert the tubes 4-5 times (avoid foam)
Take tubes on ice to 4C fridge and clamp them in the rotator for 30 minutes
Place the labeled tubes in the refrigerated centrifuge
Spin at 4C for 10 minutes at 12000 rpm
Transfer the supernatant (400 ul) to a micro tube marked TOTAL and place on ice.
From each TOTAL extract transfer 10 ul into a new micro tube and keep on ice until Part 3
Add 20 ul from the TOTAL to a new micro tube with 2 ul of Storage Buffer. Label properly.
Mix and put immediately on bucket labeled dry ice.
2. Affinity Purification
Transfer 370 ul of TOTAL from Part 1 to the tube containing IgG and label
Mix by inv