Week of 9.16 D. Ficus Chemical Transformation

 With the electroporation machine shock pod down, and the funds lacking to replace it we changed our focus to chemical transformation. The goal, similar to the electroporation goal, is to insert the pRAD1 vector into D. Ficus via chemical transformation

Here we attempt what I am calling the Tuohy Transformation protocol, a method described by Dr. Tuohy that uses simple chemical transformation methods to perform the procedure. 

This involves incubating our cells in R2B media for 24 hours at 30C, this should have been 37C.

Cells were pelleted and resuspended in fresh R2B(100ul)

40uL of CaCl2 in added and mixed

This is then incubated for 15 minutes on ice. With delays in the lab, such as lines to the stations, the cells sat for around 45 minutes in ice

8ul of pRAD1(368ng) elution was added to 30ul of culture mix

This was then incubated at 37C for 90 minutes.

1.5ml of R2B was not added tho, as the protocol called for. instead more cells were added.

This was then placed back on ice the next day and then had 1ml of R2B was added and left to incubate for another 24 hours in the shaking incubator.

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