Team:Caltech/Week 6

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Monday 7/19

Digest test gel with US and DS digested bricks. (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.

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

Digest test gel with upstream, downstream, and backbone digested bricks. (7/22)
CPCR gel featuring L17, L18, L25, & L27. (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.

Friday 7/23

CPCR gel featuring L16, L17, L18, & L21. (7/23)
CPCR gel featuring L25, L26, L27, & L29. (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

CPCR gel featuring L26 & L21. (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).
 
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