Template:SBB-Protocols inVivoGateway

From OpenWetWare
Jump to navigationJump to search

Day One

  • thaw an aliquot of gateway cells on ice and add 10uL of KCM to cells (there are currently 90uL in there right now, so you will be bringing them to a total volume of 100uL)
  • transform 1uL of your entry plasmid (basic part in pBca9523) into 30uL of the mixture of cells and KCM (standard ice for 10 minutes, heat shock followed by 2 minute cold shock)
  • rescue at 37C with shaking for 15-30 minutes
  • grow 75-130uL in 3-5uL of media (with Spec and the two assembly vector antibiotics) overnight with shaking at 37C

Day Two

  • miniprep cultures and retransform into pir-righty or pir-lefty cells and plate on appropriate double antibiotic plate (not on Spec)

Day Three

  • restreak on spec plates when picking colonies to make sure that the you have not co-transformed the entry vector with the desired product

Day Four

  • miniprep cultures and digestion map to make sure that the correct product was made
  • proceed with assembly