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