Template:SBB-Protocols inVivoGateway
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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
