Transformations

What you will need:

• “Competent” XL10 Gold cells (100 uL per transformation)
• 1 uL of plasmid you want to transform
• 1 mL of LB (non-contaminated) per transformation
• Plate with correct antibiotics (3 per transformation)

Procedure:

• Obtain XL10 Gold* cells from the –70 C freezer (GXXX, grey Nunc freezer, 2nd shelf from bottom) and allow to thaw on ice. Aliquots are about 200 ul. Take out only what you will use.
• Pipet 1uL of the plasmid (or 5 ul of ligation reaction) you want to transform into an Eppendorf tube. Add MilliQ water to 10 ul.
• When XL10 cells are thawed, pipet 100 uL into the tube with the plasmid. Pipet up and down to mix gently. Place tube on ice 30 minutes.
• After incubating on ice for 30 minutes, place cells in incubator set to 42 C for 90s. (Use the water bath near the front sink in G308 – its temperature will be most accurate. This step must be done for EXACTLY 90 s).
• Return the tube to ice for 2 min.
• Add 1mL (1000uL) of LB to the tube.
• Incubate at 37 C for 20-30 min.
• Spread cells on plate with appropriate antibiotic: plate 200ul, 20ul in 80ul of LB broth, and 2ul in 98ul of LB broth. Let dry.
• Place plates inverted in incubator at 37C overnight.

Notes

• The competent cells we have made up are XL10-Gold – if we need to transfer them to DH5alpha at a later date we can retransform the final BioBricks
• Can try 200 ul of cells instead of 100 ul to attempt to increase efficiency of transformation.