Aim: Transformation Lab

1. Aim: Transformation Lab Do now: Get into lab groups. Do not touch materials

2. Step 1 • Mark one 15 sterile 15 mL tube + Plasmid • Mark one 15 sterile 15 mL tube - Plasmid Step 2 • Use sterile pipette to add 250µL of Ice cold Calcium Chloride to each tube Step 3 • Put both tubes on ice

3. Step 4 • Use a sterile plastic loop to transfer E. Coli from starter plate to tube labeled + Plasmid Step 5 • Suspend cells by repeadtedlypipetting in and out with sterile pipet. • Should appear milky white

4. Step 6 • Return +plasmid tube to ice and repeat steps 4-5 with –plasmid tube Step 7 • Return -plasmid tube to ice • Both tubes are now in ice

5. Step 8 Step 9 • Use another sterile loop to add a loopfull of plasmid to +plasmid tube • Return Plasmid + tube to ice • Time 15 mins Step 10 • During 15 mins label : • 1 LB/amp plate plasmid + • 1 LB/amp plate plasmid – • 1LB plate plasmid + • 1LB plate plasmid -

6. Step 11 • Heat shock by imersing tubes in 42*C water bath for 90 seconds Step 12 • Use sterile pipette to add 250µL of Buria broth to each tube. • Gently tap tubes to mix LB with cell suspension. • Place tubes back on rack

7. Step 13 • Remove some cells from each tube and spread them over the plates according to your labels Step 14-16 • Use a sterile pipet to add 100µLfrom each tube to its appropriate plate • Clam shell plate and pour 4-6 glass beads onto each plate • Move plates in a back and forth motion to evenlly spread cells • Let plates rest for 5 mins • To remove beads hold plate in clamp shell over empty beaker and tap beads into empty beaker

8. Step 17 • Wrap plates together using tape • All upside down • Put group name on tape

9. Streaking a plate

10. pVIB Plasmid