User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/21

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  • pLLX8 PCR amplification product for cycloheximide counterselection was not optimized (buffer G actually sucks pretty badly). Ran a panel of failsafe buffers and found that buffers A and B yield the cleanest and most amount of PCR product for recombinational cloning.
  • Selection for capture product from lysed (NaOH) yeast cells (colony PCR) indicates that 23/24 randomly selected clones on Uracil Cycloheximide (2.5) plates were successfully captured and recombined.
  • Engineering of pBC 436 to remove oriT is underway after realizing that dual OriT's will screw things up severely downstream. (SpeI is sufficient to release the construct!!!! XhoI digest is not necessary!!!)