Team:Northwestern/09 19

Notebook

Monday, September 19th

Tasks:

Paul

  • Submitted sequencing:
    • 22-27: TorA m2 through TorA m4 (two of each, e.g. m2.1=22, m2.2=23)
    • 28-33: YcdO m2 through m4
    • 34-39: AmiA m2 through m4
  • Received sequencing for TorA: Pretty decent, most of them look good. Some point mutations on some

Sara

  • Restriction digest of cas9 and pSB1C3 using NEB protocol for the cas9 and the iGEM protocol for pSB1C3
    • Cas9:
      • 0.5 uL Ecori-HF
      • 0.5 uL SpeI
      • 2.5 uL Cutsmart
      • 19.5 uL H20
    • pSB1C3
      • Made the master mix of:
        • 0.5 uL Ecori-HF
        • 0.5 uL SpeI
        • 5 uL Cutsmart
        • 0.5 uL BSA
        • 0.5 uL Dpn1
        • 19 uL H20
      • 4 uL of MM
      • 4 uL pSB1C3 backbone
    • Incubated for 1 hr at 37°C
    • Heat inactivated for 20 min at 80°C
    • Put in freezer for tomorrow

Tasfia

  • Made sequencing reactions
  • DpnI digested Cas9 insert for pET28a
  • DpnI digested pET28a PCR purified product; heat killed enzyme
  • Ran gel screen on failed ligations and Cas9 insert for pET28a
  • Ran gel screen on Cas9-SS and Cas9-ClyA PCR products for pET28a
  • Ran Cas9-ClyA for pET28a PCR (One 50-μL reaction)
    • 1 μL template (Cas9-ClyA Gibson miniprep labeled “ClyA,” 73.9 ng/μL) (7.4 ng)
    • 1 μL 10 μM Cas9_pET28a_insertFWD primer
    • 1 μL 10 μM Pet28_ClyA_REV primer
    • 1 μL DMSO
    • 21 μL nuclease-free water
    • 25 μL OneTaq 2X Master Mix with Standard Buffer
  • DpnI digested Cas9-SS products
  • Sent DeLisa culture to Kelly so we can make comp cells out of them tomorrow
  • Jordan and Sara PCR purified the Cas9-SS for pET28a and Cas9 for pET28a
  • Sent for sequencing:
    1. FhuD Cas9 Gibson
    2. INP Cas9 1 Gibson
    3. INP Cas9 2 Gibson
    4. AmiA Cas9 1 Gibson
    5. AmiA Cas9 2 Gibson
    6. ClyA Cas9 1 Gibson
    7. ClyA Cas9 2 Gibson
    8. YcdO Cas9 1 Gibson
    9. YcdO Cas9 2 Gibson
    10. NapA Cas9 1 Gibson
    11. NapA Cas9 2 Gibson
    12. TorA m1-1 GG
    13. TorA m1-2 GG
    14. TorA m5-1 GG
    15. TorA m5-2 GG
    16. YcdO m1-1 GG
    17. YcdO m1-2 GG
    18. YcdO m5-1 GG
    19. YcdO m5-2 GG
    20. AmiA m1-1 GG
    21. AmiA m1-2 GG

Tyler

  • PCR of all SS +Cas9
    • Used annealing temp of 52°C (5 cycles) 60°C
    • Used TorA 8, DsbA 1, Ycdo 2, ClyA 1
    • 1µL template
    • 1 µL fwd primer
    • 1 µL rev primer
    • 21 µL water
    • 25 µL master mix

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