Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/01

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Here are the transformation results from last week's digestion and ligation of DBN001 (gBlock) insert into the pSB1A3 vector, replacing GFP:

  • negative control: no colonies.
  • positive control: numerous colonies, but non-fluorescent under UV light.
  • sample: no colonies.

Could be a wrong choice of plasmid, or something went wrong during the digestion/ligation. Will repeat the process after doing a test digest on the plasmid to make sure the cut sites are there. Should see about using replacement plasmid, maybe one with RFP instead of GFP, as that's easier to visualize.

DBN002 f2 and f3 primers have arrived, as well as gBlock for and rev primers. To do tomorrow:

  1. PCR on gBlock using primers. Run a gel to check size when done, check concentration on Take3 plate.
  2. PCR on DBN002 fragment from gel extraction. Make 2 products: DBN002 f2 and DBN002 f3. Use DBN002 r1 for both. Temperature gradient on both. Check annealing temp. of primers before running. Run a gel to check size of products when done.
  3. Restriction digest on EsaI/GFP plasmid from last week's digest/ligation. Use EcoRI and SpeI. Run a gel to check size of products when done, compare to René's notes on expected sizes. Or find it on Benchling somewhere.
  4. Make more pSB143-GFP plasmid and KAH187/MV2 plasmid, starting with transformation of E. coli cells. 3 plates: 2 samples and one negative control.