12:30pm Kyliah

We came in and found that across all six plates (total of 700μL plated), there were only 6 successfully transformed (RFP-dyed red) colonies, and one white colony that looked like there may have been a stop mutation in the RFP region of the plasmid.

Possible causes of low transformation efficiency:

- Insufficient suspension before drawing off for plating - make sure we suspend properly or draw from the bottom

- Bad cells - not likely, they worked just fine with the pUC plasmid

- Diluted the plasmid too far - there was that dye colour in there, may have confounded the nanospec; since the BioBrick protocol says to use 1-2μL of rehydrated plasmid, maybe next time we'll try a 1/1 or 1/10 dilution instead of 1/100

- Water used to rehydrate plasmid - we could use TE next time

We chose four parts to Rehydrate and Transform:

We chose to use 2μL of plasmid rehydrate this time, because the 2-20μL pipette reaches to the bottom of the glass test tubes we're using.

We plated two plates of each, one with 100μL and one with 50μL; the remainders in the tubes went in the fridge.

The, uh... The mCherry 100μL plate was poured a little thin, so while I was spreading, it, uh, accidentally broke a chunk of gel in the centre. We stuck it in the incubator anyway, the periphery should be fine.

We also started a broth culture of the RFP-generator transformant from yesterday.

(back to Lab notebook)