Lab 8 Procedure

Extract Yeast Genomic DNA from Yeast Culture

• Working in a group of 2, label one microfuge tube with the colony number.
• Add 1.5 mL of the colony culture into the tube.
• Spin down the tube in a microcentrifuge at 500g for 2 minutes.
• Remove the supernatant as much as you can.
• Add 120 μL of YD Digestion Buffer into the tube.
• Resuspend the pellet by vertexing and incubate at 37 °C for 20 minutes.
• Add 120 μL of YD Lysis Buffer. Mix well by gentle vertexing.
• Add 250 μL of chloroform. Mix thoroughly for 20 seconds.
• Centrifuge the tube at 12,000g for 2 minutes (be sure the centrifuge is balanced).
• Load the supernatant onto the column and centrifuge at 12,000g for 1 minute.
• Add 300 μL of DNA Washing Buffer and centrifuge at 12,000g for 1 minute. Dispose of the flow through.
• Add another 300 μL of DNA Washing Buffer and centrifuge at 12,000g for 1 minute. Dispose of the flow through.
• Re-centrifuge the tube at 12,000g for 30 seconds.
• Remove the top portion of the column (that contains that filter) and put it into a new microfuge tube.
• Add 30 μL of elution buffer (EB) to the center of the column and incubate for 1 minute at room temperature.
• Place the column/tube into the centrifuge.
  • You will not be able to cap the tube due to the column. Place the column/tube in a way that the cap of the tube is the closest to the center of the centrifuge.
• Spin for 1 minute at 12,000g in the centrifuge.
• To maximize yield, pipette the 30 μL solution back to the column (that still contains s small amount of your PCR product) and re-spin for 1 more minute at 12,000g.

Transformation of 10G E. Coli with the purified pRS315-T7 vector via electroporation 

• Thaw two aliquots of 10G E. Coli cells on ice.
• Place two cuvettes on ice.
• Add 2 μL of pRS315-T7 vector to the one tube of the 10G cells (experimental tube).
• Add 2 μL of EB to the other 10G tube (control tube).
• Label both tubes well.
• Mix by gently flicking the tubes.
• Incubate the tubes on ice for 1 minute.
• Add the cell/vector content to the chilled cuvette and tap the mixture to the bottom of the cuvette.
• Put the cuvette back on ice.
• Using the electroporator, put the cuvette in the chamber slide securely.
• Press the pulse button.
• Take the cuvette out of the chamber and add 1 mL of SOC immediately to the cuvette.
• Repeat the transformation process with the control tube.
• Transfer the solution to two new microfuge tubes.
• Shake the tube in a 37°C shaker for recovery for 20 minutes.

Phage virion formation/bacterial lysis

• During recovery, obtain two tubes of the 5 mL LB and label it with your names, group number, section number, and control or mutant.
• After 20 minutes, add 600 μL of outgrowth (bacteria after recovery) to each of the 5 mL LB tubes.
• Incubate the tube at 37°C shaker at 250rpm overnight.
• The phages will make phage particles and kill the bacteria during the incubation. You will procure the phage particles/virions next time.