Lab July 29 2009

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: Part 	Registry no. 	  Location double transcription stop 	B0015 	plate 1, 23L mCherry CDS 	J06504 	plate 1, 13F λcI promoter 	R0051 	plate 1, 6K ribosome binding site (standard) 	B0034 	plate 1, 2M

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.

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