Jordan

• PCR’d GFP 1-4 and mCherry 1-5
• 25 uL One Taq MM
• 1 ul DMSO
• 1 ul 10 mM fwd primer (Sara diluted these)
• 1 ul 10 mM rev primer
• 1 ul template (~1 ng)
• 21 uL water
• Conditions:
• Initial denaturation, 95C/3:00
• 5 cycles- 95/:30, 54/0:10, 72/0:43
• 25 cycles- 95/:30, 68/0:10, 72/0:43
• 72/5:00

Michelle

• PCR for Cas9 lnrz ClyA
• 18.5 uL water
• 1 uL DMSO
• 0.5 uL Cas9 1:3 miniprep
• 2.5 uL Tet lnrz for Cas9 fwd (10 uM)
• 2.5 uL ClyA rev (10 uM)
• 25 uL 2X Q5 polymerase MM
• Conditions: 95°C (3:00) | 95°C (0:30), 65°C (0:15), 72°C (2:40) | 72°C (5:00)
• Cam plates:
• 350 mL LB + 5.2528 g Bacto Agar
• LB:
• 10.0494 g tryptone
• 5.0101 g yeast extract
• 5.0015 g NaCl
• 1 mL 1M NaOH
• H₂O to 1L

Paul

• Submitted Sequencing:
• 5 uL of DNA for all Cas9-Dsba’s (~450 ng)
• 10 uL of DNA for all gRNA’s (~200-450 ng)
• Tubes “1-12”= Cas9-Dsba 1-10, A, B
• Tubes “13-22”= gRNA 1-10

Sam

• Followed up on Field museum
• Caught up OneNote
• To do: Auburn Gresham powerpoint
• To do (eventually): redesign survey

Shu

• PCR of INP
• Ran gel screening
• Went over what people have done

Tasfia

• PCR: Linearized tet backbone for GFP (same conditions and reaction mix as 08.29.16)
• Two 50-uL reactions 45 ng (“Tet-Lrz-GFP A”)
• 4.5 ng (“Tet-Lrz-GFP B”)
• PCR: Adding homology to INP (re-running because on first run forgot to DpnI digest before purifying)
• One 50-uL reaction
• 1 uL INP miniprep template (8.6 ng)
• 1 uL 10 uM INP_fwd primer
• 1 uL 10 uM INP_rev primer
• 1 uL DMSO
• 21 uL nuclease-free water
• 25 uL OneTaq 2X Master Mix with Standard Buffer
• One INP PCR product—DpnI digested
• PCR purified Tet-Lrz-GFP, Cas9-Lrz-ClyA, and [not-DpnI-digested] INP
• Tet-Lrz-GFP A: 20.1ng/uL, 260/280: 1.93, 260/230: 1.46
• Tet-Lrz-GFP B: 18.8ng/uL, 260/280: 1.99, 260/230: 2.49
• Cas9-Lrz-ClyA: 125.2ng/uL, 260/280: 1.91, 260/230: 2.06
• Questionable INP: 119.4ng/uL, 260/280: 1.89, 260/230: 1.94
• PCR: Cas9-Res-Dig (eventually for parts submission)
• One 50-uL reaction
• 1 uL “Cas9 1:3” miniprep template (13 ng)
• 1 uL 10 uM forward primer
• 1 uL 10 uM reverse primer
• 1 uL DMSO
• 21 uL nuclease-free water
• 25 uL OneTaq 2X Master Mix with Standard Buffer
• DpnI (0.5 μL enzyme) digested for ~2 hours at 37°C, then left at room temperature overnight
• Ran gel screen on unpurified, DpnI-digested Cas9-Res-Dig product



• DpnI digesting (0.5 uL enzyme) at room temperature overnight
• GFP 1-4 for golden gate
• mCherry 1-5 for golden gate
• INP with added homology (second INP PCR product of the day)
• Cas9-Res-Dig (even though it was already going on the heat block for two hours before)

Tyler

• Finished sequencing reactions, sent them off
• Inoculated 5mL cultures of TorA-Cas9