“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.