Team:Caltech/Week 6

Monday 7/19

• Group meeting!
  • Discussed issues with cloning and next week's focus.
• Performed a digest (now for 2hr) and ligations (L18-23). Transformed into DH5α.
• Turns out that the sequencing primers we ordered (G00100 & G00101) are incorrect - they appear to bind on the wrong sides of the insert. We ordered new primers designed to bind to the BioBrick prefix and suffix.
• Ran gel of digest product to verify correct bands.

Digest test gel with US and DS digested bricks. (7/19)

Tuesday 7/20

• All ligation transformations failed. Will try 16hr ligations and purifying digest products with a PCR purification kit.
• Made new growth curves testing AIBN effect on cells in LB
• Performed another live/dead assay
  • Had solutions of cells in glycerol and AIBN
  • Plated cells every hour for three hours
  • Combined glycerol solutions with LB and plated the solution
• New 16hr ligation procedure:
  • 30μL total: 2μL T4 ligase, 3μL 10x ligase buffer, 5μL vector, 10μL of each brick. Incubate for 16hr at 16°C.
• Started more cultures to miniprep tomorrow: I13504, J04450, K274002, M30109.

Wednesday 7/21

• Transformed 3μL of each ligation product from yesterday (time constants all w/in 3.8-4.0). Plated 50μL of each on plates.

Thursday 7/22

• Prepared new sequencing primers in preparation for CPCR.
• Transformation plates are almost completely empty - will centrifuge and plate remaining transformation cells.
• Colony PCR mixture:
  • 10μL 10x PCR buffer, 2μL dNTP mix, 1μL forward primer, 1μL reverse primer, 0.5μL Taq polymerase, fill to 100μL total. (Optional: add 20μL Qiagen Q buffer. Doesn't appear to help any of our reactions.)
  • Pick colony and add to water (85.5μL). Incubate at 100°C for 10min. Add remaining ingredients and begin PCR program:
    • Initial denaturation: 94°C, 4min
    • 30 cycles:
      • 94°C, 30s
      • 54°C, 30s
      • 72°C, 2min (1min/kb)
    • Final extension: 72°C, 10min
    • Finish: 5°C forever
• Ran colony PCR on 4 ligations, 2 had the correct insert length: L17, L27.
• Ran gel of digest products from yesterday to verify correctness of the digestion.

Digest test gel with upstream, downstream, and backbone digested bricks. (7/22)

Friday 7/23

• Plates containing the centrifuged transformation cells contained many colonies!
• Began 21 more CPCR reactions on ligations: L17, L18, L21, L27, L29, L16, L26, L25
  • Note that because L18 is a coding sequence, it required the coding prefix primer; all others used the noncoding prefix primer.
• Received our heat shock promoter from the Registry today! Made a plate and culture of this brick (K112400).
• Purified yesterday's 4 CPCR reactions using a PCR purification kit.
  • Concentrations of each are all quite low (<20ng/μL) - we may want to increase the number of PCR cycles.
• Ran 2 gels of all the CPCR results:

Weekend 7/24-25

Saturday 7/24

• Started 4 new CPCR reactions on L21 and L26 to verify them more accurately.
  • Disposed of all failed cultures...
  • Prepped successful clones: 17-6, 18-3, 21-3, 26-1, 27-4, 29-2, and the heat shock promoter (K112400).
• Prepared digest:
  • K215000 (u/d)
  • K112400 (u/d)
  • B0034 (u)
  • R0082 (u)
  • L17 (u)
  • L18 (u/d)
  • L26 (u)
  • I13504 (d)
  • L21 (d)
• Began ligations: L16, L19, L42, L43, L44, L23, L24, L31, L40, L34, L35
• Ran another gel of the CPCR products:

Sunday 7/25

• Transformed all ligations from yesterday (time constants all within 3.6-4.0). Used 4μL for all, except for L34 & L35, which sparked, so used 3μL instead.
• Made 2YT for making more electrocompetent cells.
• Plated all transformation cells (centrifuged at 1000g for 15min).